Homogenous populations of molecules

ABSTRACT

The invention provides populations of molecules that are prepared as, or treated to become, homogeneous for one or more molecular characteristics. In an aspect, the invention relates to molecular weight standards that may be used to determine the molecular weight or apparent molecular weight of uncharacterized molecules, such as proteins and nucleic acids, as well as in other applications. In one aspect, the molecular weight standards are pre-stained.

RELATED APPLICATION DATA

This application is a continuation and claims the right of priorityunder 35 U.S.C. §120 to U.S. application Ser. No. 12/861,764, filed Aug.23, 2010, now abandoned, which is a continuation of U.S. applicationSer. No. 10/949,471, filed Sep. 24, 2004, now U.S. Pat. No. 7,781,173,which claims the benefit of priority under 35 U.S.C. §119(e)(1) of U.S.Ser. No. 60/506,410, filed Sep. 25, 2003 and U.S. Ser. No. 60/582,209,filed Jun. 22, 2004 all of which are commonly owned with the presentapplication and the entire contents of which are hereby expresslyincorporated by reference in their entirety as though fully set forthherein.

FIELD OF THE INVENTION

The invention relates to populations of molecules that are prepared as,or treated to become, at least partially homogeneous for one or moremolecular characteristics.

BACKGROUND OF THE INVENTION

The term “molecule” is, of course, known in the art. However, it isimportant to note that the word “molecule” encompasses two concepts.First, a molecule can be the mental picture of a specific molecule as astructure that may be represented by a chemical formula or sequence.Second, the thing that is or comprises a specific molecule, i.e., apopulation of (about 100 or more) molecules. For example, 18 g of watercomprises 6.023×10²³ (Avogadro's number) molecules of H₂O.

Although a molecule in the former sense is devoid of other components, acomposition comprising an actual population of a given molecule may alsocomprise other molecules (e.g., contaminants). Populations of moleculesare said to be “pure” or to have been “purified” if the number of typesor amounts of contaminants have been removed or depleted fromcompositions comprising populations of specific molecules.

It should be noted that even a pure population of molecules can beheterogeneous with regards to one or more characteristics. For example,as is described in more detail below, a specific protein isglycosylated, i.e., many amino acid residues in the protein havecarbohydrates chemically attached thereto. However, in a population ofglycosylated proteins, some proteins may be completely glycosylated (allpossible glycosylation sites have been glycosylated in all proteins),whereas some may be only partially glycosylated proteins (i.e., onlysome of the possible glycosylation sites have been glycosylated in allprotein and/or not all of proteins are glycosylated to the same extent).In addition, the glycan structures may not be the same on all theglycation sites on the protein.

The invention provides homogeneous, or nearly homogeneous, populationsof a molecule that have advantages over heterogeneous populations of thesame molecule. Such molecules are useful as, for example, molecularstandards.

Compositions and methods useful for the elucidation of the primarystructure (i.e., sequence) of an uncharacterized protein or nucleic acidis useful in many applications in fields such as molecular biology,biotechnology, informatics, genomics and proteomics. In order toidentify and/or characterize a previously unknown and/or uncharacterizedmolecule, molecular standards are used.

A “molecular standard” is a molecule that is used to determine acharacteristic of an unknown and/or on-test molecule in an assay, suchas an analytical method. For example, a molecular standard can be aprotein that is used as a molecular weight marker in protein gelelectrophoresis, as illustrated in the Examples provided herein.

A “set of standards” are compositions that comprises (i) two or moreknown molecules differing in at least one detectable characteristicand/or (ii) two or more containers having different concentrations of aknown molecule. As a simple example of the latter type of molecularstandard, a set of solutions is prepared as a set of serial dilutions ofa solution having a known concentration of a known molecule. Although itis customary to have more than one molecule in a molecular weightstandard to allow the molecular weight of an unknown molecule to beestimated by interpolation, a single reference standard may be usefulfor comparison directly with an unknown sample. Differing mobility orother property between the unknown and the standard will indicate lackof identity in the molecular weight or other property being assessed.

The set of standards can be used with an assay that changes in aconcentration-dependent way to estimate the concentration of themolecule in the test sample, or to calibrate the settings of a device ormachine that measures a characteristic of the molecule. A comparison ismade of the signal from the unknown molecule to signals from knownmolecules using a variety of techniques known to those skilled in theart. By way of non-limiting example, proteins and nucleic acids areanalyzed using techniques such as electrophoresis, sedimentation,chromatography, and mass spectrometry.

For example, one basic characteristic of a molecule is its molecularweight (MW). Comparison of an uncharacterized molecule to a set ofstandards of known and different molecular weights (often called amolecular weight “ladder”) allows a determination of the apparentmolecular weight of the uncharacterized molecule.

For example, proteins, nucleic acids and other molecules areelectrophoresed or subjected to high pressure liquid chromatography(HPLC) in order to determine basic characteristics thereof. The MW of anuncharacterized protein can be estimated using HPLC, or electrophoresis,such as polyacrylamide electrophoresis (PAGE), including SDS-PAGE, andother techniques known in the art.

A variety of protein and nucleic acid molecular weight standards arecommercially available. However, the molecular weight standards may notcorrespond closely enough in size to the unknown sample protein to allowan accurate estimation of apparent molecular weight. Moreover, some ofthe standards give poorly resolved (e.g., “fuzzy”) bands. Some are notuseful for hybridization techniques (Southerns, Northerns, Westerns,etc.) because they do not transfer well to nitrocellulose or PVDFmembranes. Others comprise co-migrating contaminants. All of theseeffects operate to reduce the precision and accuracy of the analyticalmethod.

Protein standards of higher MWs (e.g., greater than from about 180 toabout 500 kD) are problematic. In addition to the potential problemsthat apply to MW standards in general, high MW proteins are hard toprepare, whether by recombinant DNA technology or otherwise. Many ofthese formed by cross-linking a single species of a protein to obtain aseries of multimers of the protein that have molecular weights that are2-fold, three-fold, etc., of the MW of the protein monomer (for example,Sigma sells cross-linked Hemoglobin, having an apparent molecular weightof 280 kDa). Cross-linkers are added by chemical reactions, and it isoften difficult to establish reaction conditions wherein multimerizationproceeds to the desired degree. Moreover, crosslinking is ordinarilyperformed using reagents that react with functional groups on amolecule. Generally there are more than one such reactive functionalgroup on a protein, so when protein molecules are crosslinked, a varietyof products results. For example, if a protein has as few as 4 reactivesites, 16 different crosslinked entities would be formed leading toinhomogeneity in the marker.

Moreover, the degree of homogeneity in a molecular population of aprotein is also affected by, among other things, the different types andextents of post-translational and other chemical modifications thereof.The modifications range from amino acid changes through to the additionof macromolecules: lipid, carbohydrate or protein. Also chemicalmodifications such as phosphorylation, alkylation, deamidation and suchcan occur. Many variants of the common amino acids can occur, which canaffect the structure or function of the protein. A major class ofmodification includes glycosylation, which may be N-linked, O-linked, orglycosylphosphatidylinositol (GPI)-linked. Such modifications have rolesin protein stability and folding, targeting and recognition. Lipidmodification of proteins (e.g., prenylation, myristoylation,GPI-anchoring, etc.) is also common. See Nalivaeva et al.,Post-translational Modifications of Proteins: Acetylcholinesterase as aModel System, Proteomics 1:735-747 (2001).

For proteins, a non-exhaustive list of exemplary protein molecularweight standards (protein molecular weight “ladders”) includes thefollowing:

The pre-stained Broad Range protein molecular weight standard (Bio-RadLaboratories, Hercules, Calif., Cat. No. 16001-018), which is composedof eight proteins:

Protein Molecular Weight (kDa) Myosin (H-chain) 209 beta-Galactosidase124 Bovine Serum Albumin 80 Ovalbumin 49 Carbonic Anhydrase 34 SoybeanTrypsin Inhibitor 29 beta-Lactoglobulin 21 Aprotinin 7.1

Protein Molecular Weight Markers, HPLC (Calbiochem):

Protein Molecular Weight (kDa) Glutamate dehydrogenase 290.00 Lactatedehydrogenase 142.00 Enolase 67.00 Myokinase 32.00 Cytochrome c 12.40

High Molecular Weight Protein Standards (Bio-Rad):

Protein Molecular Weight (kDa) Myosin 200.00 beta-Galactosidase 116.25Phosphorylase B 97.40 Serum Albumin 66.20 Ovalbumin 45.00

Molecular weight markers, ¹⁴C-methylated For Molecular Weights14,300-220,000 (Sigma M8932), which is a mixture of six ¹⁴C-methylatedproteins:

Protein Molecular Weight (kDa) Myosin 220.00 Phosphorylase b 97.40Albumin, Bovine Serum 66.00 Ovalbumin 46.00 Carbonic Anhydrase 30.00Lysozyme 14.30

Molecular weight markers, ¹⁴C-methylated For Molecular Weights2,350-30,000 (Sigma M8807), which is a mixture of five ¹⁴C-methylatedproteins:

Protein Molecular Weight (kDa) Carbonic Anhydrase 30.00 Soybean TrypsinInhibitor 21.50 Cytochrome c 12.50 Aprotinin  6.50 Insulin (Bovine) 5.74* *After sample preparation, the bovine insulin will probablymigrate as insulin a chain (2.35 kDa) and insulin b chain (3.40 kDa)

PEPPERMINTSTRICK™ phosphoprotein molecular weight standards (MolecularProbes, P-33350):

Protein Molecular Weight (kDa) beta-Galactosidase 116.25 Albumin, BovineSerum 66.20 Ovalbumin 45.00 Beta-Casein 23.60 Avidin 18.00 Lysozyme14.40

A need exists for homogeneous populations of molecules having a knownvalue for a molecular characteristic. Such populations can be comparedto an uncharacterized molecule in order to estimate or determine thepresence or absence of, or value for, a molecular characteristic.

A need exists for sets of molecules (molecular standards) having a knownvalue for a molecular characteristic, such as molecular weight, whereinthe value for the molecular characteristic is precise. A particular needexists for protein standards (protein “ladders”) that comprise proteinshaving a high molecular weight.

Patents and Published patent applications of Interest:

U.S. Pat. No. 5,449,758 (Protein Size Marker Ladder).

U.S. Pat. No. 5,580,788 (Use of Immunoglobulin-Binding ArtificialProteins as Molecular Weight Markers).

U.S. Pat. No. 5,714,326 (Method for the Multiplexed Preparation ofNucleic Acid Molecular Weight Markers and Resultant Products).

U.S. Pat. No. 5,578,180 (System for pH-neutral Longlife PrecastElectrophoresis Gel).

U.S. Pat. No. 6,514,938 and published U.S. Patent ApplicationUS/2002/0115103 (Copolymer 1 Related Polypeptides for use as MolecularWeight Markers and for Therapeutic Use).

Published PCT patent application WO 02/13848 and published U.S. PatentApplication US/2002/0155455 (Highly Homogeneous Molecular Markers forElectrophoresis).

SUMMARY OF THE INVENTION

The present invention provides a purified population of macromoleculesthat have been treated by the addition, removal or modification ofchemical groups, so as to be made at least partly homogeneous, for oneor more molecular characteristics. The macromolecules are typicallymolecular standards that can be nucleic acids or proteins. In certainaspects of the invention, the molecular standards are polypeptides, suchas oligopeptides and that are part of a protein, or the molecularstandards are nucleic acids, such as a DNA, RNA or oligonucleotides.

In certain aspects, the population of macromolecules is a population ofprotein standards.

In one embodiment, the present invention provides an isolated highmolecular weight protein standard. The protein standard typically has anapparent molecular weight of at least 300 kDa by SDS PAGE, and can havean apparent molecular weight, for example, of 400 kDa, 450 kDa, 500 kDa,600 kDa, 700 kDa, 750 kDa, or 1000 kDa.

In certain aspects, the isolated high molecular weight protein standarddoes not include at least one post-translational modification that isusually present on the protein in vivo. For example, the absence of apost-translational modification can be the result of chemicalmodification of the protein carried out according to a method providedherein.

In illustrative embodiments, the molecular weight standard is achemically modified high molecular weight protein. The isolated highmolecular weight protein standard in certain aspects does not include aphosphate group, a carbohydrate group, an amide, an N-terminalmodification, an isoprenoid group, a selenium group, a sulfate group, adisulfide bond, a fatty acid group, a hydroxyl group, and/or a ubiquitingroup.

In certain aspects, the protein standard is a laminin polypeptide. Incertain examples, the laminin polypeptide does not include acarbohydrate group or a disulfide bond, and optionally includes an alkylgroup.

In certain aspects, the isolated high molecular weight protein standardis at least partially homogeneous, or completely homogeneous.

In another embodiment, provided herein, is a method for preparing aprotein standard, comprising modifying an isolated protein by removing,adding, or modifying a chemical modification of the isolated protein toproduce a modified protein, thereby preparing the protein standard.

In certain illustrative examples, the chemical modification of theisolated protein is a post-translational modification. Furthermore, incertain illustrative examples, the modification of the isolated proteinis carried out for a population of isolated proteins to produce an atleast partially homogeneous population of the protein standard, or acompletely homogeneous population of the protein standard, for one,many, or all molecular characteristics. Additionally, the method can becarried out for a series of isolated proteins having different molecularcharacteristics. For example, the method can be carried out for a seriesof isolated proteins having different molecular weights.

The method can be carried out, for example, for a population of each ofa series of isolated proteins having different molecular characteristicsresulting in a series of at least partially homogeneous populations ofproteins, or a series of completely homogeneous populations of proteins.The series of isolated proteins, for example, can be a series of highmolecular weight proteins, such as high molecular weight standards. Theseries can also include other molecular weight standards, that forexample, are of a lower molecular weight.

In illustrative examples, the at least partially homogeneous populationof proteins is a population of high molecular weight proteins. Forexample, the population of proteins can include a laminin polypeptide.

The removing, adding, or modifying in certain aspects of the inventionincludes adding, removing, or modifying from an isolated protein, aphosphate group, a carbohydrate group, an amide, an N-terminalmodification, an isoprenoid group, a selenium group, a sulfate group, afatty acid group, a hydroxyl group, or an ubiquitin group. In oneexample, a phosphate group is removed from the population of isolatedproteins, to produce an at least partially homogeneous population of theprotein standard, or a completely homogeneous population of the proteinstandard, especially with respect to the presence of the group beingadded, removed, or modified.

In certain aspects, a population of a laminin polypeptide is modified.For example, the modification can include deglycosylation, reduction,and/or alkylation. The invention also provides an isolated molecularweight standard produced according to the methods provided herein.

In another embodiment, provided herein is a kit comprising an isolatedhigh molecular weight standard produced using the methods providedherein. The kit can further include a molecular weight protein standardthat is not a high molecular weight protein standard.

In certain aspects, the kit further includes software for using theisolated high molecular weight standard to determine the molecularweight of an on-test protein. The uncharacterized and/or on-testmolecule can be a polypeptide, an oligopeptide, or a protein, or can bea nucleic acid, such as DNA, RNA or an oligonucleotide.

In another embodiment, the present invention provides a set of molecularstandards that comprises (a) one or more molecules having at leastone-known value for at least one molecular characteristic and (b) atleast one homogeneous population of the one or more molecules. Invarious embodiments, homogeneous populations of molecules are preparedby adding, deleting or changing one or more chemical modifications tothe molecules to produce a population of modified molecules. In someembodiments, the chemical modifications are post-translationalmodifications of proteins. The methods of preparing homogeneouspopulations of molecules can optionally comprise purifying or partiallypurifying modified molecules and/or eliminating or partially eliminatingmolecules that have not been modified as desired.

The present invention further provides a method of using a molecularstandard to estimate or determine one or more characteristics of anuncharacterized and/or on-test molecule, wherein the molecular standard(a) comprises one or more molecules having at least one known value forat least one molecular characteristic and (b) comprises at least onehomogeneous population of a molecule, wherein the method comprisessubjecting the molecular standard and the uncharacterized molecule to acondition, and comparing the molecular standard to the uncharacterizedand/or on-test molecule.

In certain aspects of the invention, the condition to which themolecular standard and the uncharacterized molecule are subjected toinvolves separating (resolving) the molecules and detecting the resolvedmolecules. Methods of separation include for example, electrophoresisand chromatography. Methods of separation can be based upon molecularweight, molecular size (e.g., Stoke's radius), charge, isoelectric point(pI) or any other molecular characteristic that can be detected,preferably quantified.

In certain methods provided herein, the modified protein is used as astandard in an analytical method. For example, the present inventionprovides molecular standards that can be used to calibrate an instrumentwith regard to one or more molecular characteristics. In someembodiments, the molecular characteristic is molecular weight (MW),which may be an apparent MW or an actual MW.

In certain aspects of the invention, the molecules in the molecularstandard are pre-stained. Furthermore, in illustrative aspects of theinvention, the a molecular standard provided herein is used as amolecular weight standard in gel electrophoresis, such as a proteinstandard for protein gel electrophoresis. The present invention alsoprovides kits that include the high molecular weight protein standardsof the present invention, or kits for carrying out the methods providedherein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the structure of an O-linkage to GalNAc. FIG. 1B shows thestructure of an N-linkage to GalNAc.

FIG. 2A shows the structure of a high-mannose family of N-linked sugarfamily. FIG. 2B shows the structure of a complex family of N-linkedsugar family. FIG. 2C shows the structure of a hybrid family of N-linkedsugar family.

FIG. 3 shows the formation of 3′phosphoadenosine 5′-phosphosulfate.

FIGS. 4A and 4B show results of electrophoretic experiments. FIG. 4A isan image of a 3-8% Tris-Acetate gel that has been electrophoresed andstained; lane 1, laminin deglycosylated with PNGase F; lane 2, untreatedlaminin. FIG. 4B is an image showing a molecular weight standardcomprising the laminin subunits (the upper three bands) that have beendeglycosylated and reduced and alkylated.

FIG. 5A shows results of electrophoretic experiments, i.e. an image of a3-8% Tris-Acetate gel loaded with HIMARK™ Pre-Stained Standard and a 460kDa kinase. FIG. 5B shows an image of a 7% Tris-Acetate gel loaded withHIMARK™ Pre-Stained Standard and a 460 kDa kinase.

FIG. 6 shows results of electrophoretic experiments, i.e. an image of a3-8% Tris-Acetate gel that has been electrophoresed; lane 1, SEEBLUE®Plus2 Pre-Stained Standard; lane 2, HIMARK™ Pre-Stained Standard.

FIG. 7 shows results of electrophoretic experiments, i.e. an image of a7% Tris-Acetate gel that has been electrophoresed; lane 1, SEEBLUE®Plus2 Pre-Stained Standard; lane 2, HIMARK™ Pre-Stained Standard.

FIG. 8 shows results of electrophoretic experiments, i.e. an image of a4% Tris-Glycine gel that has been electrophoresed; lanes 1 and 2,HIMARK™ Pre-Stained Standard; lane 3, SEEBLUE® Plus2 Pre-StainedStandard.

FIG. 9 shows results of electrophoretic experiments, i.e. an image of a4-12% NUPAGE® gel that has been electrophoresed with 1×MES; lane 1,SEEBLUE® Plus2 Pre-Stained Standard; lane 2, HIMARK™ Pre-StainedStandard.

FIG. 10 shows results of electrophoretic and transfer experiments, i.e.an image of a 3-8% Tris-Acetate gel that has been electrophoresed, andthe samples western transferred onto a nitrocellulose membrane; lane 1,HIMARK™ Pre-Stained Standard; lane 2, SEEBLUE® Plus2 Pre-StainedStandard.

FIG. 11 shows results of electrophoretic and transfer experiments, i.e.an image of a 7% Tris-Acetate gel that has been electrophoresed, and thesamples western transferred onto a nitrocellulose membrane; lane 1,HIMARK™ Pre-Stained Standard; lane 2, SEEBLUE® Plus2 Pre-StainedStandard.

FIG. 12 shows results of electrophoretic, transfer and western blottingexperiments, i.e. an image of an 8% Tris-Acetate gel that has beenelectrophoresed, western transferred onto a nitrocellulose membrane, andwestern blotted with specific polyclonal antibody to apolipoproteinB-100 from human plasma (referred to herein as “large human protein”);lane 1, large human protein (arrow); lane 2, HIMARK™ Pre-StainedStandard; lane 3, SEEBLUE® Plus2 Pre-Stained Standard.

FIG. 13 shows results of electrophoretic, transfer and western blottingexperiments, i.e. an image of an 8% Tris-Acetate gel that has beenelectrophoresed, western transferred onto a nitrocellulose membrane, andwestern blotted with anti-6×His antibody; lane 1, BENCHMARK™ UnstainedStandard; lane 2, HIMARK™ Pre-Stained Standard; lane 3, SEEBLUE® Plus2Pre-Stained Standard.

FIG. 14 shows results of electrophoretic and transfer experiments, i.e.an image of a 3-8% Tris-Acetate gel that has been electrophoresed, andthe samples western transferred onto a PVDF membrane in the presence of20% methanol and 1× Antioxidant (Catalog No. NP0005, Invitrogen,Carlsbad, Calif., USA); lane 1, HIMARK™ Pre-Stained Standard; lane 2,SEEBLUE® Plus2 Pre-Stained Standard.

FIG. 15 shows results of electrophoretic and transfer experiments, i.e.an image of a 7% Tris-Acetate gel that has been electrophoresed, and thesamples western transferred onto a PVDF membrane in the presence of 20%methanol and antioxidant; lane 1, HIMARK™ Pre-Stained Standard; lane 2,SEEBLUE® Plus2 Pre-Stained Standard.

FIG. 16 shows results of electrophoretic, transfer and western blottingexperiments, i.e. an image of a 3-8% Tris-Acetate gel that has beenelectrophoresed, western transferred onto a PVDF membrane, and westernblotted with specific polyclonal antibody to large human protein; lane1, large human protein (arrow); lane 2, HIMARK™ Pre-Stained Standard;lane 3, SEEBLUE® Plus2 Pre-Stained Standard.

FIG. 17 shows results of electrophoretic, transfer and western blottingexperiments, i.e. an image of a 7% Tris-Acetate gel that has beenelectrophoresed, western transferred onto a PVDF membrane, and westernblotted with anti-6×His antibody; lane 1, BENCHMARK™ Unstained Standard;lane 2, HIMARK™ Pre-Stained Standard; lane 3, SEEBLUE® Plus2 Pre-StainedStandard.

FIG. 18 shows results of electrophoretic and transfer experiments, i.e.an image of a 4% Tris-Glycine gel that has been electrophoresed, and thesamples western transferred onto a nitrocellulose membrane; lane 1,SEEBLUE® Plus2 Pre-Stained Standard; lane 2, HIMARK™ Pre-StainedStandard.

FIG. 19 shows results of electrophoretic and transfer experiments, i.e.an image of a 4-12% Bis-Tris gel run with MOPS that has beenelectrophoresed, and the samples western transferred onto anitrocellulose membrane; lane 1, HIMARK™ Pre-Stained Standard; lane 2,SEEBLUE® Plus2 Pre-Stained Standard.

FIG. 20 shows results of electrophoretic experiments, i.e. an image of a3-8% Tris-Acetate gel that has been electrophoresed and stained withSIMPLYBLUE™ Safe Stain; lane 1, fibronectin; lane 2, large humanprotein, lane 3, human kinase; lane 4, HIMARK™ Pre-Stained Standard.

FIG. 21 shows results of electrophoretic experiments, i.e. an image of a3-8% Tris-Acetate gel that has been electrophoresed and stained withSYPRO® Orange; lane 1, HIMARK™ Unstained Standard; lane 2, human kinase;lane 3, large human protein; lane 4, fibronectin; lane 5, HIMARK™Pre-Stained Standard.

FIG. 22 shows results of electrophoretic experiments, i.e. an image of a3-8% Tris-Acetate gel that has been electrophoresed and stained withSYPRO® Ruby; lane 1, HIMARK™ Unstained Standard; lane 2, human kinase;lane 3, large human protein; lane 4, fibronectin; lane 5, HIMARK™Pre-Stained Standard. The number of ug of sample added in lanes 1-4 inFIG. 22 are 10% of that loaded in FIG. 21.

DETAILED DESCRIPTION

Please note: The headings used herein are for convenience only.

Contents

General Discussion of the Invention

I. Protein Modifications

A. Glycosylation

B. Cross-Linking Modifications

C. C-terminal Modifications

D. N-terminal Modifications

E. Phosphorylation

F. Methylation/Prenylation

G. Selenoproteins

H. Sulfation

I. Fatty Acid Modifications

J. Other Types of Protein Modifications

II. Changing the Type and/or Amount of Protein Modification

A. In Vivo Methods of Modifying Proteins

B. In Vitro Methods of Modifying Proteins

-   -   1. Chemically Modifying Proteins    -   2. Enzymatically Modifying Proteins        III. Detection and Analysis of Modified Proteins

A. In General

B. Amidation

C. Glycosylation and Deglycosylation

D. Phosphorylation

IV. Applications

A. Electrophoresis

B. Capillary Electrophoresis (CE)

C. High Throughput Screening (HTS).

D. Kits

E. Pre-Stained High Molecular Weight Markers

The present invention is based in part on the discovery that an improvedmolecular standard can be produced by modifying a population of aspecies of isolated molecular standards, to further modify, add, orremove chemical groups on the molecular standards, to make the isolatedmolecular standards at least partially homogeneous for one or moremolecular characteristics.

The present invention provides a purified population of macromoleculesthat have been treated by the addition, removal or modification ofchemical groups, so as to be made at least partly homogeneous, for oneor more molecular characteristics. The macromolecules are typicallymolecular standards that can be nucleic acids or proteins. In certainaspects of the invention, the molecular standards are polypeptides, suchas oligopeptides and that are part of a protein, or the molecularstandards are nucleic acids, such as a DNA, RNA or oligonucleotides.

In certain aspects, the population of macromolecules is a population ofprotein standards.

In one embodiment, the present invention provides an isolated highmolecular weight protein standard. The protein standard typically has anapparent molecular weight of at least 250 kDa by SDS PAGE, and can havean apparent molecular weight, for example, of 300 kDa, 350 kDa, 400 kDa,450 kDa, 500 kDa, 600 kDa, 700 kDa, 750 kDa, or 1000 kDa.

In certain aspects, the isolated high molecular weight protein standarddoes not include at least one post-translational modification that isusually present on the protein in vivo. For example, the absence of apost-translational modification can be the result of chemicalmodification of the protein carried out according to a method providedherein. In certain aspects, the protein standard is pre-stained (i.e.dye conjugated or dye-labeled before being analyzed by a proteinseparation technique).

In illustrative embodiments, the molecular weight standard is achemically modified high molecular weight protein. The isolated highmolecular weight protein standard in certain aspects does not include aphosphate group, a carbohydrate group, an amide, an N-terminalmodification, an isoprenoid group, a selenium group, a sulfate group, adisulfide bond, a fatty acid group, a hydroxyl group, and/or a ubiquitingroup.

In certain aspects, the protein standard is a laminin polypeptide. Incertain examples, the laminin polypeptide does not include acarbohydrate group or a disulfide bond, and optionally includes an alkylgroup. In other examples, the high molecular weight protein standard isapolipoprotein B or human kinase (Sigma, St. Louis, Mo.).

In certain aspects, the isolated high molecular weight protein standardis at least partially homogeneous, or completely homogeneous.

In certain aspects, the isolated high molecular weight protein standardis at least partially homogeneous, or completely homogeneous.

A “molecular characteristic” is any property of a molecule or populationof molecules that can be detected and/or measured. The most fundamental“molecular characteristic” of a molecular species is its presence orabsence in a composition. The invention provides compositions andmethods to detect the absence or presence of a homogeneous population ofmacromolecules. By way of non-limiting example, a homogeneous populationof macromolecules may be used to confirm that macromolecules and othermolecules are electrophoresing, flowing or otherwise moving through ananalytical or preparative media. Because of its homogeneous state, sucha population of macromolecules provides a sharp signal as it is detectedas it moves into, through or out of the media. A homogeneous populationof macromolecules can be used in such embodiments even if it is the onlymolecular species and/or no molecular characteristic other than thepresence or absence thereof is known.

A molecular characteristic may, but need not, be one that can be used todistinguish one molecular species from another. The molecularcharacteristic may, but need not, be one that can be detected and/ormeasured in other molecular species. The molecular characteristic may,but need not, be one that can be measured (either qualitatively orquantitatively) for both a known (previously characterized)macromolecule and an uncharacterized molecule. In the latter instance,the presence, absence, or amount of the signal from a knownmacromolecule can be compared with that of an uncharacterized molecule,in order to determine the presence or absence of the uncharacterizedmolecule, and/or to estimate or determine a value for the molecularcharacteristic of the uncharacterized molecule.

By “homogenous or partly so,” “homogeneous or partly so,” “at leastpartly homogeneous,” or “at least partly homogenous,” it is meant thatthe population of macromolecules is homogeneous, nearly homogeneous,homogeneous to a detectable limit, and/or homogeneous to the extentneeded to be used to practice the invention. In certain aspects, thepopulation of macromolecules, such as a population of protein standardsis at least 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100% (i.e.completely) homogeneous. By “homogeneous” or “homogenous” is meant thatthe population of macromolecules is identical with respect to theirmolecular structure. In other words, if a protein is ordinarilyglycosylated, a population of the protein likely includes individualprotein molecules that include different numbers of carbohydrate groupsand/or different types of carbohydrates. However, if the glycosylatedprotein is made homogeneous using the methods disclosed herein, thepopulation is completely or 100% homogeneous with respect toglycosylation when all of the molecules of the protein in the populationhave identical carbohydrate moieties attached at identical residues, orlack a carbohydrate moiety altogether. Therefore, the population can behomogeneous with respect to a particular characteristic (e.g.,glycosylation), or can be homogeneous with respect to allcharacteristics.

The macromolecules may be polymers, including polymers from naturalsources, such as proteins and nucleic acids. In some embodiments whereinthe macromolecule is a protein, the modification may be related to apost-translational modification such as the presence, absence, orquantity of phosphate groups, carbohydrate groups, amides, N-terminalmodifications, isoprenoid groups, selenium groups, sulfate groups,disulfide bonds, fatty acid groups, hydroxyl groups, and/or ubiquitingroups.

The present invention further provides molecular standards that may beused to estimate or determine one or more characteristics of anuncharacterized molecule, wherein the molecular standard comprises twoor more molecules, at least one of which is homogeneous. In someembodiments, the molecular characteristic is molecular weight (MW),which may an apparent MW or an actual MW. In one embodiment, the presentinvention provides one or more high molecular weight standards. Forexample, the invention provides a set of molecular weight standardswherein at least one standard is greater 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, or 1000 kDa. An exemplary set of molecularweight standards is the HIMARK™ standard set illustrated in the Examplesherein.

By “high molecular weight” protein standard it is meant that the actualor apparent MW is greater than or equal to about 200 kDa to about 10,000kDa, e.g., ≧about 250 kDa, ≧about 300 kDa, ≧about 350 kDa, ≧about 400kDa, ≧about 450 kDa, ≧about 500 kDa, ≧about 550 kDa, ≧about 600 kDa,≧about 65.0 kDa, ≧, about 700 kDa, ≧about 750 kDa, ≧about 800 kDa,≧about 850 kDa, ≧about 900 kDa, ≧about 950 kDa, ≧about 1,000 kDa, ≧about2,000 kDa, ≧about 5,000 kDa, ≧about 7,500 kDa, and ≧about 10,000 kDa,etc.

In another embodiment, provided herein, is a method for preparing aprotein standard, comprising modifying an isolated protein by removing,adding, or modifying a chemical modification of the isolated protein toproduce a modified protein, thereby preparing the protein standard.

In certain illustrative examples, the chemical modification of theisolated protein is a post-translational modification. Furthermore, incertain illustrative examples, the modification of the isolated proteinis carried out for a population of isolated proteins to produce an atleast partially homogeneous population of the protein standard, or acompletely homogeneous population of the protein standard, for one,many, or all molecular characteristics. Additionally, the method can becarried out for a series of isolated proteins having different molecularcharacteristics. For example, the method can be carried out for a seriesof isolated proteins having different molecular weights.

The at least partially homogeneous population of molecules providedherein, is typically substantially pure. “Substantially pure” refers toa population of molecules that is substantially free of other biologicalmolecules such as nucleic acids, proteins, lipids, carbohydrates orother materials with which it is naturally associated. Substantiallypurified or “isolated” refers to molecules, either nucleic or amino acidsequences, that are removed from their natural environment, isolated orseparated, and are at least 60% free, preferably 75% free, and mostpreferably 90% free from other components with which they are naturallyassociated. One skilled in the art can isolate molecules using standardtechniques for biomolecular purification. A substantially pure or purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel. An “isolated” or “purified” molecule is at least 90%free from other components with which it is naturally associated.

The method of the invention can be carried out, for example, for apopulation of each of a series of isolated proteins having differentmolecular characteristics resulting in a series of at least partiallyhomogeneous populations of proteins, or a series of completelyhomogeneous populations of proteins. The series of isolated proteins,for example, can be a series of high molecular weight proteins, such ashigh molecular weight standards. The series can also include othermolecular weight standards that for example, are of molecular weightsthat are lower than the high molecular weight standards.

In illustrative examples, the at least partially homogeneous populationof proteins is a population of high molecular weight proteins that canbe standards. For example, the population of proteins can include one ormore laminin polypeptides. For example, the laminin polypeptide can be alaminin alpha chain, a laminin beta chain, and/or a laminin gamma chain.In certain aspects, the invention provides a molecular weight standardset that includes an at least partially homogeneous population oflaminin alpha chain, laminin beta chain, and laminin gamma chain. Themolecular weight standard set is useful, for example, for determiningthe molecular weight for an on-test and/or unknown protein run on thesame gel as the protein standard. The laminin alpha protein can have anestimated molecular weight, for example, of 425-525 kDa, 475-525, or inone example 500 kDa. The laminin beta protein can have an estimatedmolecular weight, for example, of 200-375 kDa, 250-325 kDa, or inillustrative example, 290 kDa. The laminin gamma protein can have anestimated molecular weight, for example, of 200-300 kDa, 225-275 kDa, orin certain aspects, 240 kDa.

The removing, adding, or modifying in certain aspects of the inventionincludes adding, removing, or modifying from an isolated protein, aphosphate group, a carbohydrate group, an amide, an N-terminalmodification, an isoprenoid group, a selenium group, a sulfate group, afatty acid group, a hydroxyl group, or an ubiquitin group. In oneexample, a phosphate group is removed from the population of isolatedproteins, to produce an at least partially homogeneous population of theprotein standard, or a completely homogeneous population of the proteinstandard, especially with respect to the presence of the group beingadded, removed, or modified.

In certain aspects, a population of a laminin polypeptide is modified.For example, as illustrated in the Examples provided herein, themodification can include deglycosylation, reduction, and/or alkylation.The invention also provides an isolated molecular weight standardproduced according to the methods provided herein.

In another embodiment, provided herein is a kit comprising an isolatedhigh molecular weight standard produced using the methods providedherein. The kit can further include a molecular weight protein standardthat is not a high molecular weight protein standard.

In certain aspects, the kit further includes software for using theisolated high molecular weight standard to determine the molecularweight of an test protein. The uncharacterized and/or on-test moleculecan be a polypeptide, an oligopeptide, or a protein, or can be a nucleicacid, such as DNA, RNA or an oligonucleotide. The kit in other aspects,include at least one of the at least partially homogeneous lamininproteins discussed herein.

In another embodiment, the present invention provides a set of molecularstandards that comprises (a) one or more molecules having at least oneknown value for at least one molecular characteristic and (b) at leastone homogeneous population of the one or more molecules. In variousembodiments, homogeneous populations of molecules are prepared byadding, deleting or changing one or more chemical modifications to themolecules to produce a population of modified molecules. In someembodiments, the chemical modifications are post-translationalmodifications of proteins. The methods of preparing homogeneouspopulations of molecules can optionally comprise purifying or partiallypurifying modified molecules and/or eliminating or partially eliminatingmolecules that have not been modified as desired.

The present invention further provides a method of using a molecularstandard to estimate or determine one or more characteristics of anuncharacterized and/or on-test molecule, wherein the molecular standard(a) comprises one or more molecules having at least one known value forat least one molecular characteristic and (b) comprises at least onehomogeneous population of a molecule, wherein the method comprisessubjecting the molecular standard and the uncharacterized molecule to acondition, and comparing the molecular standard to the uncharacterizedand/or on-test molecule.

In certain aspects of the invention, the condition to which themolecular standard and the uncharacterized molecule are subjected toinvolves separating (resolving) the molecules and detecting the resolvedmolecules. Methods of separation include electrophoresis andchromatography. Methods of separation can be based upon molecularweight, molecular size (e.g., Stoke's radius), charge, isoelectric point(pI) or any other molecular characteristic that can be detected,preferably quantified.

In certain methods provided herein, the modified protein is used as astandard in an analytical method. For example, the present inventionprovides molecular standards that can be used to calibrate an instrumentwith regard to one or more molecular characteristics. In someembodiments, the molecular characteristic is molecular weight (MW),which may be an apparent MW or an actual MW.

In certain aspects of the invention, the molecules in the molecularstandard are pre-stained. Furthermore, in illustrative aspects of theinvention, the molecular standard provided herein is used as a molecularweight standard in gel electrophoresis, such as a protein standard forprotein gel electrophoresis.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

I. Protein Modifications

The following sections provide details regarding various proteinmodifications, which are examples of chemical modifications with respectto the present invention. These modifications can be affected in themethods provided herein in order to provide a more homogeneous proteinpopulation.

I.A. Glycosylation

Glycosylation at an -Asn-Xaa-Cys- site has been reported for coagulationprotein C. N-linked sites are often indirectly assigned by theappearance of a “blank” cycle during sequencing. The oligosaccharide canbe released by treatment with Peptide N Glycosidase F (PNGase F,available from Prozyme, San Leandro, Calif.), which converts theglycosylated Asn to Asp.

Glycoproteins consist of proteins covalently linked to carbohydrate. Thepredominant sugars found in glycoproteins are glucose, galactose,mannose, fucose, N-Acetyl-D-galactosamine (GalNAc),N-Acetyl-D-glucosamine (GlcNAc), and NANA (N-acetylneuraminic acid).

The carbohydrate modifications found in glycoproteins include, by way ofnon-limiting example, carbohydrates that are linked to the proteincomponent through either O-glycosidic or N-glycosidic bonds. TheN-glycosidic linkage is through the amide group of asparagine. TheO-glycosidic linkage is to the hydroxyl of serine, threonine orhydroxylysine. The linkage of carbohydrate to hydroxylysine is generallyfound only in the collagens. The linkage of carbohydrate to5-hydroxylysine is either the single sugar galactose or the disaccharideglucosylgalactose. In Ser- and Thr-type O-linked glycoproteins, thecarbohydrate directly attached to the protein is GalNAc. In N-linkedglycoproteins, it is GlcNAc. FIG. 1A illustrates the structure of anO-linkage to GalNAc, and FIG. 1B illustrates the structure of anN-linkage to GlcNAc.

The predominant carbohydrate attachment in glycoproteins of mammaliancells is via N-glycosidic linkage. The site of carbohydrate attachmentto N-linked glycoproteins is found within a consensus sequence of aminoacids, N—X—S(T), where X is any amino acid except P (proline). N-linkedglycoproteins all contain a common core of carbohydrate attached to thepolypeptide. This core consists of three mannose residues and twoGlcNAc. A variety, of other sugars are attached to this core andcomprise three major N-linked families (1) the high-mannose type (FIG.2A), which contains all mannose outside the core in varying amounts; (2)the hybrid type (FIG. 2C), which contains various sugars and aminosugars; and (3) the complex type (FIG. 2B), which is similar to thehybrid type, but in addition, contains sialic acids to varying degrees.

I.B. Cross-Linking Modifications

Cystine (—CH₂—S—S—CH₂—) disulfides spontaneously form under oxidizingconditions; lanthionine (—CH₂—S—CH₂—) has also been found, often as anartifact of peptide synthesis. Intramolecular thioester linkages betweenCys and Glu residues have been found in complement C3 and C4, and inalpha-2-macroglobulin. Epsilon-(gamma-glutamyl)lysine cross-links arecatalyzed by transglutaminases such as factor XIIIa. UbiquitinC-terminal COOH are similarly linked to Lys epsilon-amino groups.

I.C. C-terminal Modifications

C-Terminal amidation is common in peptide hormones. The amide iscontributed by Gly from a precursor C-terminal sequence of —XGXX.

I.D. N-terminal Modifications

N-acetyl “blocked” N-termini of eukaryotic proteins are common; theN-terminal residues are often Ala, Ser, Met, Gly or Thr. N-acetylresidues can be enzymatically removed from peptides (Krishna, R. G.(1992) in: Techniques in Protein Chemistry 111, pp. 77-84, AcademicPress (San Diego). N-methylation of mammalian ribosomal proteins usuallyoccurs at Ala/Pro/Phe-Pro-Lys-N-termini. N-formyl Met is usuallyprocessed by a deformylase. Glutamine and S-carboxy-methylcysteine canform cyclic “blocked” N-terminal residues; the former can be removed bypyroglutamate aminopeptidase.

Some proteins have the 14 carbon myristoyl group added to theirN-termini. The donor for this modification is myristoyl-CoA.

I.E. Phosphorylation

Post-translational phosphorylation is one of the most common proteinmodifications that occurs in animal cells. It is estimated that ⅓ of allproteins present in a mammalian cell are phosphorylated and thatkinases, enzymes responsible for phosphorylation, constitute about 1-3%of the expressed genome. The vast majority of phosphorylations occur asa mechanism to regulate the biological activity of a protein and as suchare transient. In other words a phosphate (or more than one in manycases) is added and later removed.

The enzymes that phosphorylate proteins are termed kinases and thosethat remove phosphates are termed phosphatases.

That is, protein kinases catalyze the reaction:ATP+Protein→Phosphoprotein+ADP

Whereas, in contrast, protein phosphatases catalyze the reaction:Phosphoprotein+H2O→Protein+P_(i)

In animal cells serine, threonine and tyrosine are the amino acidssubject to phosphorylation. The largest group of kinases are those thatphosphorylate either serines or threonines and as such are termedserine/threonine kinases. The ratio of phosphorylation of threedifferent amino acids is approximately 1000/100/1 forserine/threonine/tyrosine. However, a phosphate group can modifyhistidine, arginine, lysine, cysteine, glutamic acid and aspartic acidresidues. However, the phosphorylation of hydroxyl groups at serine(90%), threonine (10%), or tyrosine (0.05%) residues are the mostprevalent.

I.F. Methylation/Prenylation

Prenylation refers to the addition of the 15 carbon farnesyl group orthe 20 carbon geranylgeranyl group to acceptor proteins, both of whichare isoprenoid compounds derived from the cholesterol biosyntheticpathway. The isoprenoid groups are attached to cysteine residues at thecarboxy terminus of proteins in a thioether linkage (C—S—C). A commonconsensus sequence at the C-terminus of prenylated proteins has beenidentified and is composed of CAAX, where C is cysteine, A is anyaliphatic amino acid (except alanine) and X is the C-terminal aminoacid. In order for the prenylation reaction to occur the threeC-terminal amino acids (AAX) are first removed and the cysteineactivated by methylation in a reaction utilizing S-adenosylmethionine asthe methyl donor. For reviews, see Rando, “Chemical biology of proteinisoprenylation/methylation”, Biochim Biophys Acta 1300:5-16. (1996); andToledo et al., “Methylation of proteins from the translationalapparatus: an overview”, Arch Biol Med Exp (Santiago) 21:219-229 (1988).

I.G. Selenoproteins

Selenium is a trace element and is found as a component of severalprokaryotic and eukaryotic enzymes that are involved in redox reactions.The selenium in these selenoproteins is incorporated as a unique aminoacid, selenocysteine, during translation. A particularly importanteukaryotic selenoenzyme is glutathione peroxidase; this enzyme isrequired during the oxidation of glutathione by hydrogen peroxide (H₂O₂)and organic hydroperoxides. A selenocysteine residue in a protein hasthe general structure:

I.H. Sulfation

Sulfate modification of proteins occurs at tyrosine residues such as infibrinogen and in some secreted proteins (e.g., gastrin). Generally,sulfate is added permanently.

The universal sulfate donor is 3′-phosphoadenosyl-5′-phosphosulphate(PAPS). The formation of PAPS is illustrated at FIG. 3.

I.I. Fatty Acid Modifications

Glycosylphosphatidylinositol (GPI) structures are found at theC-terminus of several membrane proteins (Ferguson, Biochem. Soc. Trans.20:243-256, 1992). Ethanolamine phosphoglycerol attached to Glu residuesgenerate “blank” sequencer cycles. Some membrane-spanning proteins havecytoplasmic Cys (or possibly Ser) residues that are acylated bypalmitate or stearate. N-myristoylation can occur on proteins withN-terminal Gly residues (Sefton et al., J. Cell Biol 104:1449-1453,1987; Grand, Biochem. J. 258:625-638, 1989) or on the epsilon-amino sidechain of Lys (Stevenson et al., Proc. Natl. Acad. Sci. 90, 7245-7249,1993). Acyl groups can be identified by GC, GC-MS analysis or by RP-HPLCafter acid hydrolysis, extraction with ether or chloroform, andmethylation. S- or O-acyl groups will be removed by base (0.1 Mmethanolic KOH, 90 min, 23° C.) or hydroxylamine (I M NH₂OH, 20 hr, pH7, 23° C.) treatment, while N-acyl groups are base- andhydroxylamine-stable and cause “blocked” N-termini. Lipoic acid groupshave also been found on Lys. Isoprenylation of Cys residues has beenreported for Ras-type proteins (Clarke, Annu. Rev. Biochem. 61:355-386,1992). Geranylgeranyl (C20) or farnesyl (C15) isoprenoids are added toCys side chains at -Cys-Aaa-Aaa-Xaa C-termini, then the Aaa-Aaa-Xaatripeptide is removed, followed by methylation of the COOH.

I.J. Other Protein Modifications

Other non-limiting example of post-translational modifications includehydroxylation, ADP-ribosylation, carboxylation, adenylation andubiquitination. For a review, see Han et al., “Post-translationalchemical modification(s) of proteins”, Int J Biochem 24:19-28 (1992).PoSt-translational bromination of tryptophan residues is also known(Jimenez et al., “Bromocontryphan: post-translational bromination oftryptophan”, Biochemistry 36:989-994 (1997)

II. Changing the Type and/or Amount of Protein Modification

The following section provides of exemplary methods that can be used toremove, add, or modify, a chemical modification present on a protein.

II.A. In Vivo Methods of Modifying Proteins

A method for making amidated peptides using a modified self-cleavingvacuolar membrane ATPase (VMA) intein expression system is described byCottingham et al., “A method for the amidation of recombinant peptidesexpressed as intein fusion proteins in Escherichia coli”, Nat Biotechnol19:974-977 (2001).

Recombinant expression of proteins in Escherichia coli may result inglycosylation. See Mironova et al., “Evidence for non-enzymaticglycosylation in Escherichia coli”, Mol Microbiol 39:1061-1068 (2001).

Genetic engineering techniques have been used to replace cysteineresidues in proteins with selenocysteine. See, for example, Koishi etal., “Production of Functional Human Selenocysteine-ContainingKDRF/Thioredoxin Reductase in E. coli”, J. Biochem. 127:977-983 (2000);and Stadtman, “Selenocysteine” (review), Annu Rev Biochem. 65:83-100(1996).

Protein production and maturation in Saccharomyces cerevisiae isdescribed by Schuster et al., “Protein expression in yeast; comparisonof two expression strategies regarding protein maturation”, J Biotechnol84:237-248 (2000).

The post-translational machinery of Pichia pastoris has allowed for theproduction of functional mammalian glycoproteins. See Crosier et al.,“New insights into the control of cell growth; the role of the AxIfamily” Pathology 29:131-135 (1997).

Methods of modulating and/or modifying post-translational processing,including N-glycosylation, in insect cells are known. See Ailor et al.,“Modifying secretion and post-translational processing in insect cells”,Curr Opin Biotechnol 10:142-145 (1999); and Jarvis et al., “EngineeringN-glycosylation pathways in the baculovirus-insect cell system”(review), Curr Opin Biotechnol 9:528-533 (1998).

II.B. In Vitro Methods of Modifying Proteins

II.B.1. Chemically Modifying Proteins

II.B.1a. Chemical Amidation

The following are representative teachings regarding chemical amidationthat can be used to practice the invention: Bradbury et al., PeptideAmidation, Trends Biochem Sci 16:112-115 (1991); and Eipper et al.,Peptide Alpha-Amidation, Annu Rev Physiol 50:333-344 (1988).

II.B.1b. Chemical Glycosylation and Deglycosylation

The following are representative teachings or compositions regardingchemical deglycosylation that can be used to practice the invention:GLYCOFREE™ Chemical Deglycosylation Kit (Prozyme, San Leandro, Calif.,USA).

II.B.2. Enzymatically Modifying Proteins

II.B.2.a. Enzymatic Amidation

The following are representative teachings regarding enzymatic amidationthat can be used to practice the invention: Wu et al., “In VitroAmidating Processing of Products Expressed by Gene Engineering”, ActaBiochemica et Biophysica Sinica (Shanghai) 32:312-315 (2000), whichdescribes recombinant rat peptidylglycine alpha-amidating monooxygenase(rPAM); Merkler, “C-terminal Amidated Peptides: Production by the InVitro Enzymatic Amidation of Glycine-Extended Peptides etc.”, EnzymeMicrob Technol 16:450-456 (1994); and Breddam et al., “Amidation ofGrowth Hormone Releasing Factor (1-29) by Serine CarboxypeptidaseCatalysed Transpeptidation”, Int J Pept Protein Res 37:153-160 (1991):

II.B.2.b. Deamination—Amidohydrolases

According to the categorical numbering system of EC (Schomburg, D. &Salzmann, M., eds. (1991) Enzyme Handbook 4 (Springer, Berlin) that usessuch properties as substrate specificity and physicochemicalcharacteristics as criteria, amidohydrolases have been divided into twomajor types: 77 were included in the EC 3.5.1 category (EC3.5.1.1-3.5.1.77), and 14 were placed under EC 3.5.2 (EC3.5.2.1-3.5.2.14).

Amidohydrolases that can be used to practice the invention includerecombinantly produced amidohydrolases, which may be enantioselective.See, e.g., Fournand et al., “Acyl transfer activity of an amidase fromRhodococcus sp. strain R312: formation of a wide range of hydroxamicacids”, Applied and Environmental Microbiology 64:2844-2852 (1998).

II.B.2.c. Glycosylation and Deglycosylation

Enzymes catalyzing the addition (O-GlcNAc transferase, OGT) and removal(O-GlcNAcase) of the N-glycosylation have been cloned and expressedusing recombinant DNA technology. These and other enzymes of thedisclosure can likewise be cloned for expression in bacterial hosts(Vosseller et al., “Nucleocytoplasmic O-glycosylation: O-GlcNAc andfunctional proteomics”, Biochimie 83:575-581, 2001).

The following are representative of glycosylases and deglycosylases thatcan be used to practice the invention:

Enzymes available from New England Biolabs (Beverly, Mass.) include:

  N-Glycosidase F (PNGase F) from Flavobacterium meningosepticumEndoglycosidase H (Endo H) Endo Hf (a protein fusion of Endo H andmaltose binding protein)

Enzymes available from Prozyme (San Leandro, Calif.):

Enzymatic Deglycosylation Kit GLYKO ® Enzymatic Deglycosylation KitGLYKO ® Deglycosylation Plus Ceramide-Glycanase from Marobdella decoraSialidase from S. pneumoniae recombinant in E. coli Sialidase from C.perfingens recombinant in E. coli Sialidase from A. ureafaciensrecombinant in E. coli Beta-N-acetylhexosaminidase from S. pneumoniaerecombinant in E. coli Alpha-Mannosidase from X. manihotis recombinantin E. coli. O-Glycanase from S. pneumoniae recombinant in E. coliEndoglycosidase-H from S. plicatus recombinant in E. coliBeta-Galactosidase from X. manihotis recombinant in E. coli.Beta-Xylosidase from A. niger Alpha-Fucosidase from X. manihotisrecombinant in E. coli. Alpha-Fucosidase from A. niger recombinant in E.coli. Chondroitinase ABC from P. vulgaris recombinant in A. nigerEndo-beta-galactosidase from Bacteroides fragiles Endoglycosidase H(recombinant) PNGase F (Chryseobacterium [Flavobacterium]meningosepticum). Endo-alpha-N-acetylgalactosaminidaseEndoglycosidase-F1 from Flavobacterium meningosepticum N-Glycanase(recombinant) Endoglycosidase-F1 from Flavobacterium meningosepticumEndoglycosidase-F2 from Flavobacterium meningosepticumEndoglycosidase-F3 from Flavobacterium meningosepticum N-GLYCANASE ™--PLUS PNGase F (recombinant) Heparinase I (Flavobacterium heparinum)Chondroitinase ABC Chondroitinase ACI Rev 29/12/96 alpha-L-Iduronidase(Human liver - recombinant) beta(1-3,4,6)-D-Glucuronidase (Bovine liver)alpha-N-Acetylglucosaminidase (Human urine - recombinant)Iduronate-2-sulfatase (Human liver - recombinant)Glucosamine-6-sulfatase (Caprine liver - recombinant) Sulfamidase (Humanliver - recombinant) Galactosyltransferase Fucosyltransferasealpha-N-Acetylgalactosaminidase (Chicken liver)beta(1-2,3,4,6)-N-Acetylhexosaminidase (Jack bean)Beta-N-Acetylhexosaminidase alpha(1-2,3,4,6)-Fucosidase (Bovine kidney)alpha(1-3,4,6)-Galactosidase (Green coffee bean) alpha-Mannosidase(Aspergillus saitoi) alpha(1-2,3,6)-Mannosidase (Jack bean) Sialidase(Arthrobacter ureafaciens) beta(1-3,4,6)-Galactosidase (Jack bean)beta(1-3,4)-Galactosidase (Bovine Testes) beta(1-4)-Galactosidase(Streptococcus pneumoniae) beta-Mannosidase (Helix pomatia) Sialidase[Neuraminidase] (Clostridium perfingens) SIALIDASE N ™ (Newcastledisease virus, Hitchner B1 Strain) SIALIDASE N ™ (recombinant)alpha(1-3,4)-Fucosidase (Almond meal) SIALIDASE V ™ (Vibrio cholerae)Sialidase I (recombinant) Sialidase (Arthobacter ureafaciens)II.B.2.d. Phosphatases

The following are representative of phosphatases that can be used topractice the invention:

Members of the serine/threonine protein phosphatase family, includingthe prototype member, protein phosphatase-1 (phosphorylase phosphatase;originally named PR enzyme). For a review, see Lee et al., PhosphorylasePhosphatase: New Horizons for an Old Enzyme, Frontiers in Bioscience4:d270-285 (1999).

Alkaline phosphatases, such as calf intestine alkaline phosphatase(Stratagene, Promega) and alkaline phosphatase from E. coli (CHIMERx,Milwaukee, Wis.).

II.B.1b(2)(v) Kinases

The following are representative of kinases that can be used to practicethe invention: members of the eukaryotic protein kinase (EPK) family,including human members (Kostichl et al., Human Members of theEukaryotic Protein Kinase Family, Genome Biology 3:research0043.1-0043.12 (2002); members of the calmodulin-protein kinasefamily; and members of the mitogen-activated protein kinase (MAPK)family.

Polypeptides that are normally not phosphorylatable can be modified torender them phosphorylatable (see U.S. Pat. No. 5,986,006).

III. Detection and Analysis of Protein Modifications and ModifiedProteins

III.A. In General

The following are representative of teaching, compositions and methodsof detecting and/or analyzing modified or unmodified proteins that canbe used to practice the invention:

-   Jaquinod et al., “Mass Spectrometric Characterisation of    Post-Translational Modification and Genetic Variation in Human    Tetranectin”, Biol Chem 380:1307-1314 (1999);-   Kuster et al., “Identifying Proteins and Post-Translational    Modifications by Mass Spectrometry” (review), Curr Opin Struct Biol.    8:393-400 (1998);-   Kuster et al., “Glycosylation Analysis of Gel-Separated Proteins”,    Proteomics 1:350-361 (2001);-   Yan et al., “Mass Spectrometric Determination of a Novel    Modification of the N-terminus of Histidine-tagged Proteins    Expressed in Bacteria”, Biochem Biophys Res Commun 259:271-282    (1999);-   Yamagata et al., “Mapping of Phosphorylated Proteins on    Two-Dimensional Polyacrylamide Gels Using Protein Phosphatase”,    Proteomics 2:1267-1276 (2002);-   Nilsson, “Analysis of Protein Glycosylation by Mass Spectrometry”,    Mol Biotechnol 2:243-280 (1994);-   Kouach et al., “Application of Electrospray and Fast Atom    Bombardment Mass Spectrometry to the Identification of    Post-Translational and Other Chemical Modifications of Proteins and    Peptides”, Biol Mass Spectrom 23:283-294 (1994);-   Han et al., “Post-Translational Chemical Modifications of    Proteins—III. Current Developments in Analytical Procedures of    Identification and Quantitation of Post-Translational Chemically    Modified Amino Acid(s) and Its. Derivatives”, Int J Biochem    25:957-970 (1993); and-   Chen et al., published U.S. Patent Application 2003/0153007    (Automated Systems and Methods for Analysis of Protein    Post-Translational Modification).    III.B. Amidation

The following are representative of teachings, compositions and methodsof detecting and/or analyzing amidation that can be used to practice theinvention: Jones et al., A Fluorometric Assay for PeptidylAlpha-Amidation Activity Using High-Performance Liquid Chromatography,Anal Biochem 168:272-279 (1988).

III.C. Glycosylation and Deglycosylation

The following are representative of teachings, compositions and methodsof detecting and/or analyzing glycosylation and/or deglycosylation thatcan be used to practice the invention:

Available from Prozyme:

  FACE ® N-Linked Oligosaccharide Profiling Kit GAG Set I AnalyticalCalibration Set GAG Set II Analytical Calibration Set Fucose LinkageAnalysis Kit Sialic Acid Linkage Analysis Kit FACE ® N-LinkedOligosaccharide Profiling Kit FACE ® Monosaccharide Composition KitFACE ® N-Linked Oligosaccharide Sequencing KitIII.D. Phosphorylation

The following are representative of teachings, compositions and methodsof detecting and/or analyzing phosphorylation that can be used topractice the invention: Kaufmann et al., Use of antibodies for detectionof phosphorylated proteins separated by two-dimensional gelelectrophoresis (review), Proteomics 1:194-199 (2001); Sickmann et al.,Phosphoamino acid analysis (review), Proteomics 1:200-6 (2001);McLachlin et al., Analysis of phosphorylated proteins and peptides bymass spectrometry (review), Curr Opin Chem. Biol. 5:591-602 (2001); andReagan et al., published U.S. Patent Application 2003/0162230 (Methodfor Quantifying Phosphokinase Activity on Proteins).

IV. Applications

The at least partially homogeneous population of macromolecules of thepresent invention can be used for a number of applications, especiallyanalytical applications, typically within biological sciences. Theapplications include, but are not limited to, electrophoresis, includingfor example gel electrophoresis and capillary electrophoresis,high-throughput screening, high pressure and low pressure liquidchromatography especially with gel filtration media, andultracentrifugation. In one illustrative embodiment, the applicationthat includes a homogenous molecular standard of the present inventionis protein gel electrophoresis, especially SDS-polyacrylamide gelelectrophoresis.

IV.A. Electrophoresis

Methods for separating (resolving) mixtures of macromolecules haveapplications such as scientific analysis (of, by way of non-limitingexample, mixtures of proteins, as occurs in the field of proteomics),preparative techniques, diagnostic methods, regulatory analysis and thelike. One non-limiting example of a method of resolving macromolecules(such as, by way of non-limiting example, nucleic acids, polypeptidesand proteins) is electrophoresis.

Electrophoresis is a preparative and/or analytical method used toseparate and characterize macromolecules. It is based on the principlethat charged particles migrate in an applied electrical field. Ifelectrophoresis is carried out in solution, molecules are separatedaccording to their surface net charge density. If carried out insemisolid materials (gels), however, the matrix of the gel adds asieving effect so that particles migrate according to both charge andsize.

The invention is exemplified herein with regards to gel electrophoresisof macromolecules for analysis, purification or other manipulationsthereof. The electrophoretic separation is performed by conventionalmethods according to the specific method, use, format or application.

The gel-based electrophoretic embodiments of the invention can becarried out in any suitable format, e.g., in standard-sized gels,minigels, strips, gels designed for use with microtiter plates and otherhigh throughput (HTS) applications, and the like. Minigel and otherformats include without limitation those described in the followingpatents and published patent applications: U.S. Pat. No. 5,578,180, toEngelhorn et al., entitled “System for pH-Neutral LonglifeElectrophoresis Gel”; U.S. Pat. Nos. 5,922,185; 6,059,948; 6,096,182;6,143,154; 6,162,338, all to Updyke et al.; published U.S. PatentApplications 20030127330 A1 and 20030121784 A1; and published PCTApplication WO 95/27197, all entitled “System for pH-Neutral StableElectrophoresis Gel”; U.S. Pat. No. 6,057,106, to Updyke et al., andpublished PCT application WO 99/37813, both entitled “Sample Buffer andMethods for High Resolution Gel Electrophoresis of Denatured NucleicAcids”; U.S. Pat. No. 6,562,213 to Cabilly et al., and published PCTapplication WO 02/18901, both entitled “Electrophoresis Apparatus forSimultaneous Loading of Multiple Samples”; and published U.S. PatentApplication 2002/0134680 A1, to Cabilly et al., and published PCTapplication WO 02/071024, both entitled “Apparatus and Method forElectrophoresis”.

Protein electrophoresis can performed in the presence of a chargeddetergent like sodium dodecyl sulfate (SDS) which coats, and thusequalizes the charges of, most proteins, so that migration depends onsize (molecular weight). Proteins are often separated in this fashion,i.e., SDS-PAGE (PAGE=polyacrylamide gel electrophoresis). In addition toSDS, one or more other denaturing agents, such as urea, can also beincluded in order to minimize the effects of secondary and tertiarystructure on the electrophoretic mobility of proteins. Such additivesare typically not necessary for nucleic acids, which have a similarsurface charge irrespective of their size and whose secondary structuresare generally broken up by the heating of the gel that happens duringelectrophoresis.

In general, electrophoresis gels can be either in a slab gel or tube gelform. For slab gels, the apparatus used to prepare them usually consistsof two glass or plastic plates with a space disposed between them bymeans of a spacer or gasket material and the apparatus is held togetherby a clamping means so that the space created is closed on three sidesand open at the top. A solution of unpolymerized gel-monomer is pouredinto the space while in its liquid state. A means of creating wells ordepressions in the top of the gel (such as a comb) in which to placesamples is then placed in the space. The gel-monomer solution is thenpolymerized and becomes a solid gel. After polymerization is complete,the comb device is removed and the gel, while still held within theplates, is then ready for use. Examples of such apparatus are well knownand are described in U.S. Pat. No. 4,337,131 to Vesterberg; U.S. Pat.No. 4,339,327 to Tyler; U.S. Pat. No. 3,980,540 to Hoefer et al.; U.S.Pat. No. 4,142,960 to Hahn et al.; U.S. Pat. No. 4,560,459 to Hoefer;and U.S. Pat. No. 4,574,040 to Delony et al. Tube gels are produced in asimilar manner, however, instead of glass or plastic plates, glasscapillary tubing is used to contain the liquid gel.

Two commonly used media for gel electrophoresis and other separationtechniques are agarose and polyacrylamide. Each of these is described inturn as follows.

IV.A.1. Agarose

Agarose is a colloidal extract prepared from seaweed. Different speciesof seaweed are used to prepare agarose; commercially available agaroseis typically prepared from genera including, but not limited to,Gracilaria, Gelidium, and Pterocladia. It is a linear polysaccharide(average molecular mass of about 12,000) made up of the basic repeatunit agarobiose, which comprises alternating units of galactose and3,6-anhydrogalactose. Agarose contains no charged groups and is thususeful as a medium for electrophoresis.

Agarose gels have a very large “pore” size and are used primarily toseparate large molecules, e.g., those with a molecular mass greater thanabout 200 kDa. Agarose gels can be prepared, electrophoresed (“run”) andprocessed faster than polyacrylamide gels, but their resolution isgenerally inferior. For example, for some macromolecules, the bandsformed in agarose gels are “fuzzy” (diffuse). The concentration ofagarose typically used in gel electrophoresis is between from about 1%to about 3%.

Agarose gels are formed by suspending dry agarose in an aqueous, usuallybuffered, media, and boiling the mixture until a clear solution forms.This is poured into a cassette and allowed to cool to room temperatureto form a rigid gel.

IV.A.2. Polyacrylamide

Acrylamide polymers are used in a wide variety of chromatographic andelectrophoretic techniques and are used in capillary electrophoresis.Polyacrylamide is well suited for size fractionation of chargedmacromolecules such as proteins and nucleic acids (e.g.,deoxyribonucleic acids, a.k.a. DNA, and ribonucleic acids, a.k.a. RNA).

The creation of the polyacrylamide matrix is based upon thepolymerization of acrylamide in the presence of a crosslinker, usuallymethylenebisacrylamide (bis, or MBA). Upon the introduction of catalyst,the polymerization of acrylamide and methylene bisacrylamide proceedsvia a free-radical mechanism. The most common system of catalyticinitiation involves the production of free oxygen radicals by ammoniumpersulfate (APS) in the presence of the tertiary aliphatic amineN,N,N′,N′-tetramethylethylenediamine (TEMED).

In the case of acrylamide, various chemical polymerization systems maybe used. For example, TEMED and persulfate may be added to providepolymerization initiation. Once the temperature becomes stable orapproaches ambient temperature, the polymerization is assumed to becomplete. If desired, an acrylamide gradient may be developed bysuccessively adding solutions with increasing amounts of acrylamideand/or cross-linking agent. Alternatively, differential initiation maybe used, so as to provide varying degrees of polymerization and thusprepare a gradient gel.

Electrophoretic gels based on polyacrylamide, are produced byco-polymerization of monoolefinic monomers with di- or polyolefinicmonomers. The co-polymerization with di- or polyfunctional monomersresults in cross-linking of the polymer chains and thereby the formationof the polymer network. As monoolefinic monomers used in the inventioncan be mentioned acrylamide, methacrylamide and derivatives thereof suchas alkyl-, or hydroxyalkyl derivates, e.g. N,N-dimethylacrylamide,N-hydroxypropylacrylamide, N-hydroxymethylacrylamide. The di- orpolyolefinic monomer is preferably a compound containing two or moreacryl or methacryl groups such as e.g. methylenebisacrylamide,N,N′-diallyltartardiamide, N,N′-1,2-dihydroxyethylene-bisacrylamide,N,N-bisacrylyl cystamine, trisacryloyl-hexahydrotriazine. In a broadersense, “polyacrylamide gels” also include gels in which the monoolefinicmonomer is selected from acrylic- and methacrylic acid derivatives,e.g., alkyl esters such as ethyl acrylate and hydroxyalkyl esters suchas 2-hydroxyethyl methacrylate, and in which cross-linking has beenbrought about by means of a compound as mentioned before. Furtherexamples of gels based on polyacrylamide are gels made byco-polymerization of acrylamide with a polysaccharide substituted tocontain vinyl groups, for example allyl glycidyl dextran as described inEP 87995. The gels used in the invention are prepared from an aqueoussolution containing 2-40% (w/w), preferably 3-25% (w/w) of the monomersmentioned above. The amount of cross-linking monomer is about 0.5% toabout 15%, preferably about 1% to about 7% by weight of the total amountof monomer in the mixture.

In addition to the initiator and monomers the reaction mixture maycontain various additives, the choice of which will depend on theparticular electrophoretic technique contemplated. Thus, for isoelectricfocusing a certain type of amphoteric compounds are added which willcreate a pH gradient in the gel during electrophoresis. Or a gradient ofbuffering compounds bearing vinyl groups can be copolymerized into thegel so as to create an immobilized pH gradient.

IV.A.3. Composite Gels

Composite gels, formed from two or more electrophoretic media, can alsobe used. Non-limiting examples of polyacrylamide-agarose compositionsinclude those described in the following non-comprehensive list: U.S.Pat. No. 5,785,832 to Chiari et al., entitled “Covalently Cross-Linked,Mixed-Bed Agarose-Polyacrylamide Matrices for Electrophoresis andChromatography”; Andrews, “Electrophoresis on Agarose and CompositePolyacrylamide-Agarose Gels”, Electrophoresis, Clarendon Press, pg.148-177 (1986); Bates et al., “Autonomous parvovirus LuIII encapsidatesequal amounts of plus and minus DNA strands” J. Virol. 49:319-324(1984); Dahlberg et al., “Electrophoretic Characterization of BacterialPolyribosomes in Agarose-Acrylamide Composite Gels”, J Mol. Biol.41:139-147 (1969); Fisher et al., “Role of Molecular Conformation inDetermining the Electrophoretic Properties of Polynucleotides inAgarose-Acrylamide Composite Gels”, Biochemistry 10:1895-1899 (1971);Horowitz et al., “Electrophoresis of Proteins and Nucleic Acids onAcrylamide-Agarose Gels Lacking Covalent Cross-Linkings”, Anal. Biochem.143:333-340 (1984); Isono et al., “Lack of ribosomal protein S1 inBacillus stearothermophilus” Proc Natl Acad Sci USA 73:767-770 (1976);Peacock et al., “Molecular Weight Estimation and Separation ofRibonucleic Acid by Electrophoresis in Agarose-Acrylamide CompositeGels,” Biochemistry 7:668-674, (1968); Rashid et al., “ElectrophoreticExtraction-Concentration of Ribonucleic Acid from Agarose-AcrylamideComposite Gels”, Anal Biochem 127:334-339 (1982); and Ringborg et al.,“Agarose-Acrylamide Composite Gels for Microfractionation of RNA”,Nature 220:1037-1039 (1968).

IV.A.4. Representative Gels

Any suitable gel and buffers can be used to practice the invention.Non-limiting examples of gels and buffers include those described hereinand in the following: U.S. Pat. No. 5,578,180, to Engelhorn et al.,entitled “System for pH-Neutral Longlife Electrophoresis Gel”; U.S. Pat.Nos. 5,922,185; 6,059,948; 6,096,182; 6,143,154; 6,162,338, all toUpdyke et al.; published U.S. Patent Applications 20030127330 A1 and20030121784 A1; and published PCT Application WO 95/27197, all entitled“System for pH-Neutral Stable Electrophoresis Gel”; U.S. Pat. No.6,057,106, to Updyke et al., and published PCT application WO 99/37813,both entitled “Sample Buffer and Methods for High Resolution GelElectrophoresis of Denatured Nucleic Acids”; U.S. Pat. No. 6,562,213 toCabilly et al., and published PCT application WO 02/18901, both entitled“Electrophoresis Apparatus for Simultaneous Loading of MultipleSamples”; Published U.S. Patent Application 20020134680 A1, to Cabillyet al., and published PCT application WO 02/071024, both entitled“Apparatus and Method for Electrophoresis”; and U.S. Pat. No. 5,785,832,to Chiari et al., entitled “Covalently Cross-Linked, Mixed-BedAgarose-Polyacrylamide Matrices for Electrophoresis and Chromatography.”

IV.A.5. Isoelectric Focusing (IEF).

One type of electrophoresis is usually referred to as isoelectricfocusing (IEF) or electrofocusing. IEF, which can be carried out in anelectrophoretic medium or in solution, involves passing a mixturethrough a separation medium which contains, or which may be made tocontain, a pH gradient or other pH function. The device or gel has arelatively low pH at one end, while at the other end it has a higher pH.IEF is discussed in various texts such as Isoelectric Focusing by P. G.Righetti and J. W. Drysdale (North Holland Publ., Amsterdam, andAmerican Elsevier Publ., New York, 1976).

The charge on a protein or other molecule depends on the pH of theambient solution. At the isoelectric point (pI) for a certain molecule,the net charge on that molecule is zero. At a pH above its pI, themolecule has a negative charge, while at a pH below its pI the moleculehas a positive charge. Each different molecule has a characteristicisoelectric point. When a mixture of molecules is electrophoresed in anIEF system, an anode (positively charged) is placed at the acidic end ofthe system, and a cathode-(negatively charged) is placed at the basic(alkaline) end. Each molecule having a net positive charge under theacidic conditions near the anode will be driven away from the anode. Asthey electrophorese through the IEF system, molecules enter zones havingless acidity, and their positive charges decrease. Each molecule willstop moving when it reaches its particular pI, since it no longer hasany net charge at that particular pH. This effectively separatesmolecules that have different pI values. The isolated molecules ofinterest can be removed from the IEF device by various means, or theycan be stained or otherwise characterized.

Some types of IEF systems generate pH gradients by means of “carrierampholytes.” These are synthetic ampholytes that often have asignificant amount of buffering capacity. When placed in an IEF device,each carrier ampholyte will seek its own isoelectric point. Because oftheir buffering capacity, many carrier ampholytes will establish a pHplateau rather than a single point. By using a proper mixture of carrierampholytes, it is possible to generate a relatively smooth pH gradientfor a limited period of time. Such mixtures are sold commercially undervarious trade names, such as Ampholine (sold by LKB-Produkter AB ofBromma, Sweden), Servalyt (sold by Serva Feinbiochemica of Heidelberg,FRG), and Pharmalyte (sold by Pharmacia Fine Chemicals AB, Uppsala,Sweden). The chemistry of ampholyte mixtures is discussed in variousreferences, such as U.S. Pat. No. 3,485,736; Matsui et al., Methods Mol.Biol. 112:211-219 (1999); and Lopez, Methods Mol. Biol. 112:109-110(1999).

In IEF in Immobilized pH gradients (IPG), amphoteric ions are forced toreach a steady-state position along pH inclines of various scopes andspans (see Righetti et al., Electrophoresis 15:1040-1043, 1994; Righettiet al., Methods Enzymol. 270:235-255, 1996; and 2-D Electrophoresisusing immobilized pH gradients—Principles and Methods, Edition AC,Berkelman, T. and T. Stenstedt, Amersham Biosciences, Freiburg, Germany,1998). In one popular version of IPG, the pH gradient is in the form ofa strip and is referred to as a “strip gel” or a “gel strip” that can beused in appropriate formats. See, by way of non-limiting example,published PCT patent applications WO 98/57161 A1, WO 02/09220 A1,published U.S. patent application US 2003/0015426 A1, and U.S. Pat. Nos.6,599,410; 6,156,182; 6,113,766; and 6,495,017.

IV.A.6. Two-Dimensional Electrophoresis

Two-dimensional (2D) electrophoresis techniques are also known andinvolve a first electrophoretic separation in a first dimension,followed by a second electrophoretic separation in a second, transversedimension. In a common 2D electrophoretic method, proteins are subjectedto IEF in a polyacrylamide gel in the first dimension, resulting inseparation on the basis of isoelectric point (pI), and are thensubjected to SDS-PAGE in the second dimension, resulting in furtherseparation on the basis of size (O'Farrell, J. Biol. Chem.250:4007-4021, 1975).

IV.A.7. Staining Gels

A typical method for staining electrophoretic media in a gel format thatcan be carried out at ambient temperature includes the steps of fixingthe gel (e.g., incubating the gel in an aqueous solution having about40% ethanol and about 10% acetic acid for about 1 hour); rinsing thefixed gel one or more times with distilled water for about 10 minutes;incubating the gel in a staining solution for about 1 hour; and washingthe gel one or more times with water or a buffer, such as one comprisingsodium phosphate at a concentration of from about 5 to about 100 mM,e.g., 5, 10, 15, 20, 25, or 50 mM, the buffer having a pH of from about6 to about 8, e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9.

IV.B. Capillary Electrophoresis (CE)

Electrophoresis also includes techniques known collectively as capillaryelectrophoresis (CE). Capillary electrophoresis (CE) achieves molecularseparations on the same basis as conventional electrophoretic methods,but does so within the environment of a narrow capillary tube (25 to 50μm). The main advantages of CE are that very small (nanoliter) volumesof sample are required; moreover, in a capillary format, separation anddetection can be performed rapidly, thus greatly increasing samplethroughput relative to gel electrophoresis. Some non-limiting examplesof CE include capillary electrophoresis isoelectric focusing (CE-IEF)and capillary zone electrophoresis (CZE).

Capillary zone electrophoresis (CZE) is a technique that separatesmolecules on the basis of differences in mass to charge ratios, whichpermits rapid and efficient separations of charged substances (for areview, see Dolnik, Electrophoresis 18:2353-2361, 1997). In general, CZEinvolves introduction of a sample into a capillary tube, i.e., a tubehaving an internal diameter from about 5 to about 2000 microns, and theapplication of an electric field to the tube. The electric potential ofthe field both pulls the sample through the tube and separates it intoits constituent parts. Each constituent of the sample has its ownindividual electrophoretic mobility; those having greater mobilitytravel through the capillary tube faster than those with slowermobility. As a result, the constituents of the sample are resolved intodiscrete zones in the capillary tube during their migration through thetube. An on-line detector can be used to continuously monitor theseparation and provide data as to the various constituents based uponthe discrete zones.

CZE can be generally separated into two categories based upon thecontents of the capillary columns. In “gel” CZE, the capillary tube isfilled with a suitable gel, e.g., polyacrylamide gel. Separation of theconstituents in the sample is predicated in part by the size and chargeof the constituents traveling through the gel matrix. This technique,sometimes referred at as capillary Gel Electrophoresis (CGE), isdescribed by HjertñÏ (J. Chromatogr. 270:1, 1983), and is suitable forresolving macromolecules that differ in size but have a constantcharge-to-mass ratio (Guttman et al., Anal. Chem. 62:137, 1990).

In “open” CZE, the capillary tube is filled with an electricallyconductive buffer solution. Upon ionization of the capillary, thenegatively charged capillary wall will attract a layer of positive ionsfrom the buffer. As these ions flow towards the cathode, under theinfluence of the electrical potential, the bulk solution (the buffersolution and the sample being analyzed), must also flow in thisdirection to maintain electroneutrality. This electroendosmotic flowprovides a fixed velocity component which drives both neutral speciesand ionic species, regardless of charge, towards the cathode. Fusedsilica is principally utilized as the material for the capillary tubebecause it can withstand the relatively high voltage used in CZE, andbecause the inner walls of a fused silica capillary ionize to create thenegative charge which causes the desired electroendosmotic flow. Theinner wall of the capillaries used in CZE can be either coated oruncoated. The coatings used are varied and known to those in the art.Generally, such coatings are utilized in order to reduce adsorption ofthe charged constituent species to the charged inner wall. Similarly,uncoated columns can be used. In order to prevent such adsorption, thepH of the running buffer, or the components within the buffer, aremanipulated.

IV.C. High-Throughput Screening (HTS)

In some embodiments, electrophoresis is carried out in formats suitablefor high-throughput screening (HTS). Preferred HTS formats, as well asother formats for other electrophoretic applications, are described in:U.S. Pat. No. 6,562,213 to Cabilly et al., and published PCT applicationWO 02/18901, both entitled “Electrophoresis Apparatus for SimultaneousLoading of Multiple Samples”; U.S. Pat. No. 6,379,516 and published U.S.Patent Application 20020134680 A1, both to Cabilly et al., and publishedPCT application WO 02/071024, all entitled “Apparatus and Method forElectrophoresis”; and U.S. Pat. Nos. 5,582,702; 5,865,974; and6,379,516, all to Cabilly et al., and published PCT applications WO96/34276 and WO 97/41070, all entitled “Apparatus and Method forElectrophoresis.”

IV.D. Kits

In some embodiments, the homogeneous populations of molecules of theinvention are prepared as solutions to be used in kits and methods suchas electrophoresis. In certain examples, such solutions are provided“ready to go”, i.e., they can be used directly in gels without furthermanipulation. Alternatively, a stock solution is provided and is dilutedto prepare a molecular standard. Moreover, the molecules of themolecular standard can be provided in separate containers that are mixedtogether in order to prepare a molecular standard.

In one embodiment, provided herein is a kit comprising an isolated highmolecular weight standard. Furthermore, the kit can include a molecularweight protein standard that is not a high molecular weight proteinstandard. For example, the kit can include protein standards that areless than 250 kDa, as illustrated in the Examples herein. In certainaspect of the invention, the protein standards are pre-stainedstandards. Furthermore, the kit can include a set of protein standards,for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20protein standards. At least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the proteinstandards can be high molecular weight protein standards.

Liquid components of kits are stored in containers, which are typicallyresealable. A preferred container is an Eppendorf tube, particularly a1.5 ml Eppendorf tube. A variety of caps may be used with the liquidcontainer. Generally preferred are tubes with screw caps having anethylene propylene O-ring for a positive leak-proof seal. A preferredcap uniformly compresses the O-ring on the beveled seat of the tubeedge. Preferably, the containers and caps may be autoclaved and usedover a wide range of temperatures (e.g., +120° C. to −200° C.) includinguse with liquid nitrogen. Other containers can be used. Kits of theinvention in certain aspects, are stored at −20C or below.

Kits of the invention can further comprise one or more sets ofinstructions.

Kits of the invention can further comprise one or more calibration orother guides that shows one or more images of the molecular standardsafter they have been subject to a condition or process of interest, Suchas gel electrophoresis. Typically, such guides will indicate a value foreach member of the molecular standard and its correlation orrelationship with a molecular characteristic. For example, a guide forelectrophoresis might show a gel on which a protein or DNA “ladder” hasbeen electrophoresed and the molecular weights corresponding to eachband in the image.

A kit of the invention that is designed to be used to calibrate aninstrument or machine may have different sets of instructions and becalled a “Calibration Kit.” Such a kit may further include reagents orother compositions for the operation of the instrument or machine duringcalibration procedures or otherwise, such as solutions and devices forflushing lines, for rinsing gel matrices, or for cleaning sensors suchas image scanners and optical or fluorescent viewers; instructions,which may be in the form of software, for running a calibration programon the instrument or machine; and the like.

IV.E. Pre-Stained High Molecular Weight Markers

In some embodiments of the present invention, molecular standards arepre-stained to facilitate their detection. “Pre-stained” markers, asused herein, refers to marker molecules that are coupled to molecules ofa detectable substance, such as dyes that are visible or fluorescent,prior to being loaded onto an electrophoretic gel or other separationmedium or apparatus. Such pre-stained markers may be detected before,during or after electrophoresis without the need for a separate stainingstep. In some embodiments, the pre-stained molecular standards arepre-stained protein standards. In some embodiments, the pre-stainedprotein standards comprise high molecular weight proteins, e.g. proteinswith molecular weights greater than 300 kDa.

In some embodiments of the present invention, one or more species ofpre-stained molecular standards, e.g. pre-stained protein standards, areincluded in a kit. Some such kits are comprised of high molecular weightpre-stained protein standards.

EXAMPLES

The following Examples are included herewith for purposes ofillustration only and are not intended to be limiting of the invention.

-   EXAMPLE 1: LAMININ POLYPEPTIDES-   EXAMPLE 2: DEGLYCOSYLATION OF LAMININ-   EXAMPLE 3: REDUCTION AND ALKYLATION OF LAMININ-   EXAMPLE 4: MASS SPECTROMETRY-   EXAMPLE 5: APPARENT MOLECULAR WEIGHT-   EXAMPLE 6: PREPARATION OF A SET OF PROTEIN STANDARDS-   EXAMPLE 7: KIT INSTRUCTIONS-   EXAMPLE 8: DEPHOSPHORYLATION-   EXAMPLE 9: DNA METHYLATION-   EXAMPLE 10: PRE-STAINED LAMININ-   EXAMPLE 11: PRE-STAINED β-GALACTOSIDASE-   EXAMPLE 12: HiMark™ PRE-STAINED STANDARDS.

A listing of some of the chemicals and reagents used in the Examples,and the suppliers of those chemicals and reagents, is as follows.

Chemical or Reagent Supplier Iodoacetamide Sigma Dithiothreitol (DTT)Sigma α-cyano-4-hydroxycinnamic acid Aldrich Acetonitrile AldrichTrypsin Promega Fibronectin Calbiochem DNA-Dependent Kinase PromegaNormal human serum Sigma Normal rat serum Sigma 3-8% Tris-Acetate (TA)Gels Invitrogen 4% Tris-Glycine (TG) Gels Invitrogen 4-12% NUPGE ® GelsInvitrogen Ammonium bicarbonate Sigma Protease Inhibitor Cocktail ICalbiochem Protease Inhibitor Cocktail III Calbiochem PNGase F NewEngland Biolabs Endo-O-Glycosidase ProZyme Laminin Natural MouseInvitrogen Sialidase A ProZyme 20% SDS Invitrogen Triton X100 Sigma 100kD Ultra-concentrators Amicon Tri-n-butylphosphine (TBP) AldrichIsopropanol Acros Dialysis membrane 12 kDa MWCO Invitrogen IodoaceticAcid Sigma

Example 1 Laminin Polypeptides

Laminin is a protein found in the basement membrane, and consists ofthree peptide chains that are linked by disulfide bonds. There areseveral different types of laminin that vary in molecular weight. See,e.g., Miner et al., The laminin alpha chains: expression, developmentaltransitions, and chromosomal locations of alpha1-5, identification ofheterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform, JCell Biol 137:685-701 (1997); Teller et al., Interactions betweenlaminin and epithelial cells in intestinal health and disease. Exp. Rev.Mol. Med. 28 Sep. 2001; and Simon-Assmann et al., The laminins role inintestinal morphogenesis and differentiation. Ann N Y Acad Sci.859:46-64 (Review) (1998).

Unless indicated otherwise, Laminin type 1 from Engelbreth-Holms-Swarm(EHS) sarcoma grown in mouse was used in the Examples. Laminin type 1consists of three polypeptide chains: alpha 1 (335,732 Da), beta 1 (194,670 Da) and gamma. 1 (173,999 Da). The protein is glycosylated andcontains many cysteines in disulfide bridges.

Commercially available laminin is sold by Invitrogen (Carlsbad, Calif.),BD Biosciences (San Jose, Calif.), Serva (Heidelberg, Del.) and othercompanies as a 800 kilodalton (kDa) to 1000 kDa protein that comprisesthree polypeptide chains. The largest chain has a molecular weight of400 kDa. A search on the Swiss-Prot database revealed that the alpha 5chain from mice has a molecular weight of 404 kilodaltons (kDa).

Example 2 Deglycosylation of Laminin

Deglycosylation: 1 mg of laminin stock solution was mixed with 6.25 μlof 20% SDS and 31.25 μl of 2 M DTT, mixed well and incubated at roomtemperature for 30 minutes. Then, 65 μl of 14% Triton X-100 was addedand the protein solution was mixed well. 5 μl PNGase F (New EnglandBiolabs) was added and the protein solution was mixed well and was leftat 4° C. for 48 to 72 hours.

Purification: 2 ml of 50 mM Tris pH=8, 0.1% SDS buffer was added to 1 mlof deglycosylated laminin solution. The protein was concentrated bycentrifugation at 3,000×g using a filter having a molecular weightcut-off (MWCO) of 100 kDa MWCO (Amicon, Beverly, Mass.) until the samplevolume was between 0.5 to 1 ml. This protocol was repeated two moretimes, with 2 ml buffer being added each time. After the lastconcentration cycle, the protein concentration was adjusted to 1 mg/mlif needed by adding 50 mM Tris, 0.1% SDS buffer to the solution.

FIG. 4A is an image of a 3-8% Tris-Acetate gel on which has beenelectrophoresed, in lane 1, laminin deglycosylated with PNGase F and, inlane 2, untreated laminin. As can be seen in FIG. 4A, the treatedlaminin shows how the laminins bands become sharper (more tightlyresolved), and that there is a shift in apparent molecular weight. Alsoin FIG. 4A, lane 1, it can be seen that the beta- (middle band) andgamma- (lowest band) chains of the laminin, which otherwise blendtogether (compare to lane 2 of FIG. 4A), have been cleanly separated.

Example 3 Reduction and Alkylation of Laminin

Reduction and Alkylation: 100 μl of 20% SDS was added to the 1 ml of 1mg/ml deglycosylated laminin solution and the sample was incubated atroom temperature for 1 hour. Then, 40 μl 200 mM tri-n-butylphosphine(TBP) in isopropanol was added and the sample was vortexed for 10seconds. Then the sample was left in a vortexer 1 hour. After theaddition of the TBP the sample became cloudy but, by the end of theincubation, the sample solution was clear. Then, 100 μl 1 M iodoaceticacid pH=7 in 25 mM phosphate was added to the protein sample, which wasincubated for 1 hour at room temperature.

Purification: 2 ml of 50 mM Tris pH=8, 1% SDS buffer was added to 1 mlalkylated laminin solution. The protein was concentrated bycentrifugation at 3,000×g using a filter having a 100 kDa MWCO (Amicon)until the sample volume was between 0.5 to 1 ml. This protocol wasrepeated two more times, with 2 ml buffer being added each time. Afterthe last concentration cycle, the protein concentration was adjusted to1 mg/ml if needed by adding 50 mM Tris, 0.1% SDS buffer to the solution.

Laminin treated as above is, in some instances, referred to hereinafteras “modified laminin.”

Example 4 Mass Spectrometry

10 μl of the reduced and alkylated sample was loaded onto SDS-PAGE gels.After the electrophoretic run, the gels were stained with SIMPLYBLUE™safe stain and then destained in water for 1 to 2 hours at roomtemperature. Portions of the gels comprising the protein bands were cutout with a razor blade and placed in 1.5 ml tubes. The gel fragmentswere destained with two additions of 500 μl of 30% acetonitrile in 25 mMammonium bicarbonate pH=8, vortexing for 15 min after each addition. Thegel bands were dehydrated with addition of 300 μl 100% acetonitrile anddried on a SPEEDVAC® concentrator system for 5 minutes. Then 10 μl 20ng/μl Trypsin in 25 mM ammonium bicarbonate was added to each gel band.The samples were incubated for 30 min at room temperature and 20 μl 25mM ammonium bicarbonate was added to each gel band and the samples wereleft at 37° C. for 16-18 hours for digestion. After digestion the liquidin the tubes was transferred to a clean 1.5 ml tube. The gel fragmentswere extracted with 100 μl of 0.1% trifluoroacetic acid (TFA) for 30minutes. A second extraction was done with 100 μl of 50%acetonitrile/0.05% TFA, and all the extracts were pooled. The extractswere dried using a SPEEDVAC® concentrator system (Savant, Holbrook,N.Y.), and 5 μl 0.1% TFA was added to each tube. Then 3 μl of eachsample was mixed with 7 μl of 10 mg/ml alpha-cyano-4-hydroxycinnamicacid (CHCA) in 50% acetonitrile/0.05% TFA. One microliter was loaded onthe MALDI target. The analyses were done using a Voyager DE STR massspectrometer (Applied Biosystems, Foster City, Calif.).

This mass spectrometry analysis by peptide mapping revealed thatInvitrogen's laminin 1 has a large alpha 1 chain with a molecular weightof 338 kDa. According to the Swiss-Prot database, a signal peptide withlength of 24 amino acids is removed after expression, so the matureprotein size is 335,732 Da The protein was identified as laminin 1 withvery high confidence over a range of coverage of the protein's aminoacid sequence, including the N-terminus and C-terminus of the protein.

Example 5 Apparent Molecular Weight

The apparent molecular weight of the modified laminin was estimatedbased on a standard curve prepared using proteins with known molecularweights. The following protein samples were loaded on each gel: Mark 12Unstained Standard, BENCHMARK™ Protein Standard, Precision Plus, Normalhuman serum, Normal rat serum, DNA-dependent Kinase, Fibronectin, andHigh Molecular Weight Unstained Standard. The molecular weight standardswere loaded directly without any preparation.

The serum samples were prepared the following way: To 750 μl centrifugetube 20 μl serum, 25 μl 4×LDS Sample buffer, 5 μl 2M DTT and 50 μl waterwere added. The samples were reduced at 70° C. for 10 min.

The DNA-dependent kinase sample was prepared by adding 8.3 μl 4×LDS and1.6 μl 2M DTT to the tube (with 25 μl protein solution) and reducing for10 min at 70° C.

Fibronectin was prepared by adding 20 μl fibronectin solution (500μg/ml), 25 μl 4×LDS, 5 μl DTT and 50 μl water to 750 μl centrifuge tubeand reducing for 10 min at 70° C.

The samples were loaded on 4% TG (Tris-Glycine), 3-8% TA (Tris-Acetate)and 4-12% NUPAGE®/MOPS gels. The gels were stained with Coomassie R250and destained with multiple washes with 8% acetic acid.

The molecular weights of the laminin polypeptide chains calculated bytheir sequence are: alpha 1-335,732 Da, beta 1-194,670 Da and gamma1-173,999 Da. However, in these experiments, laminin polypeptide chainsalkylated with iodine acetic acid protein runs as if it had a higherapparent molecular weight. The gamma 1 chain runs larger than myosin(224 kDa) on a 3-8% TA gel when alkylated with iodine acetic acid. Theother laminin chains also seem to run as larger apparent molecularweight bands.

Without wishing to be bound by any particular theory, this is probablydue to the addition of very large number of negative charges to theprotein when alkylated with iodine acetic acid. There are 163 cysteinesin the alpha chain, 127 cysteines in the beta chain and 99 cysteines inthe gamma chain of the laminin. When the protein is alkylated withiodine acetic acid, the corresponding number of negative charges areadded to each polypeptide chain. It is possible that the highlynegatively charged molecule binds different number of SDS molecules andhas different hydrodynamic radius. Therefore it migrates differently toits true molecular weight. A change of the migration rate was notobserved for the polypeptide chains of laminin when the protein isalkylated with iodoacetamide, probably because the charge of the proteinhas not changed much, if at all, when alkylated with non-ionicalkylation reagent.

In order to establish the apparent molecular weight with which thisprotein migrates in specific electrophoretic conditions, the followingexperiments were carried out.

The estimation of the molecular weight of proteins is normally done byconstructing a calibration curve based on proteins with known molecularweights and calculating the molecular weight of the unknown proteinbased on that curve. Since there are no existing molecular weightstandards with suitable molecular weights, several proteins from humanand rat serum with molecular weight in the desired range wereidentified.

One protein from human serum—apolipoprotein B-100 precursor—wasidentified and has a molecular weight of 516 kDa according to theprotein sequence in the Swiss-Prot database (Accession #P04114).

According to the Swiss-Prot database, a signal peptide with length of 27amino acids is removed after expression, and the final molecular weightof the mature protein is 512 kDa. The protein identification was donemultiple times with high confidence of the identification and welldistributed peptide coverage map that spans from the N-terminus tonearly the C-terminus of the protein and confirmed that the identity ofthe complete (unprocessed) version of the protein.

Also identified was one protein from rat serum: rat plectin, having amolecular weight of 533 kDa (Accession #P30427). A peptide coverage mapwas prepared and shows that the identified peptides cover the sequencefrom the first few residues to nearly the C-terminus of the protein.

A commercially available protein—human DNA-dependent kinase—waspurchased from Promega (Madison, Wis.). According to the sequence ofthis protein found at the Swiss-Prot database (Accession #P78527) themolecular weight of the largest subunit of the protein is 469 kDa.

Rat fibronectin was purchased from Calbiochem (San Diego, Calif.).According to the sequence information obtained from the Swiss-Protdatabase (Accession #P04937), the molecular weight of the protein wascalculated to be 269 kDa after cleavage of 32 amino acids signalpeptide.

These four high molecular weight proteins—rat plectin (533 kDa), humanapolipoprotein B-100 precursor (516 kDa), human DNA-dependent kinase(469 kDa), and rat fibronectin (269 kDa)—together with other molecularweight standards as described herein, were run (electrophoresed) on 4%TG, 3-8% TA, 7% TA and 4-12% NUPAGE®/MOPS gels with 1×MOPS runningbuffer.

After running on SDS-PAGE gels, the Rf values of all resolved bands fromeach molecular weight standard (Mark 12, Magic Mark, Bench Mark andPrecision Plus), rat fibronectin, apolipoprotein B-100, rat plectin andhuman DNA-dependent kinase were measured using the Alpha Innotechimaging system. The molecular weights of the proteins and the Rf valuesmeasured on the Alpha Innotech imaging system were entered in an Excelspreadsheet and the log₁₀ (MW) values were calculated. Scatter plots oflog₁₀ MW v/s Rf were prepared and trendlines with R2 closest to 1 wereselected.

The log₁₀ MW values of the laminin polypeptide chains were calculatedusing the equations describing the trendline (x=Rf, y=log₁₀ MW). Themolecular weight (MW) of the polypeptide chains were calculated usingthe equation: MW=10^(y).

The apparent molecular weight of the alpha chain of laminin wascalculated to be 490 kDa. This corresponds well with the migration ofthe alpha chains on the gels (i.e. higher than the DNA-dependent kinaseand just below the apolipoprotein B-100). Close examination of themigration of the beta and gamma chains of the laminin reveals similarsituation. The calibration of these polypeptide chains is also moreaccurate (representing better the migration of the protein on the gelrelative to standards with close molecular weight). Therefore, thismethod for calibration of the laminin polypeptide chains was used on allof the gel types that were tested.

Using the trendline equations from the corresponding charts themolecular weights of the laminin bands were calculated and are asfollows:

Polypeptide 4% TG 3-8% TA 7% TA 4-12% NuPAGE Laminin 515 kDa 507 kDa 490kDa 448 kDa alpha Laminin beta 365 kDa 298 kDa 280 kDa 226 kDa Laminin272 kDa 247 kDa 227 kDa 220 kDa gamma

Although the molecular weights of the laminin polypeptide chains are:alpha 1 (335,732 Da), beta 1 (194,670 Da) and gamma 1 (173,999 Da) theapparent molecular weights (the molecular weight that these polypeptidesmigrate with on electrophoresis gel) are different.

Example 6 Preparation of a Set of Protein Standards

The following proteins were used for the preparation of a set of proteinstandards (a.k.a. as a “protein ladder”) that is sometimes hereinafterreferred to as the Unstained High Molecular Weight Standard (UHMWS).

The set of standards comprises the proteins listed in the followingtable:

Apparent Molecular Weight Protein (kDa) Laminin - alpha chain 500Laminin - beta chain 290 Laminin - gamma chain 240 160 kDa recombinant160 protein B-galactosidase 116 Phosphorylase-b 97 BSA 66 GDH 55  40 kDarecombinant 40 protein

All proteins except laminin are stock solutions from the MARK12™ orBENCHMARK™ standards (Invitrogen, Carlsbad, Calif.) (Flynn et al.,Protein Analysis with the BENCHMARK™ Protein, Focus 19:33-35 (1997),incorporated herein by reference in its entirety).

An image showing the molecular weight standard, comprising the lamininsubunits (the upper three bands) that have been deglycosylated andreduced and alkylated, after electrophoresis in a 3-8% Tris-Acetate geland staining is shown in FIG. 4B.

The CF (Concentration Factor—the amount of stock solution needed to make1 ml of Unstained High Molecular Weight Standard) of modified lamininwas determined by running dilutions of the concentrated stock solutionside by side with pre-qualified high molecular weight standard on 3-8%TA gel. The dilution that matches the bend intensity of the standard wasused to determine the CF factor. The CF factors of other proteins weretypically determined when the stock solutions were prepared.

All proteins were blended according to their CF factors and the neededvolume water and NuPAGE® LDS Sample buffer was added to achieve 1×concentration. The final formulation of the standard was stored at −20°C.

Example 7 Kit Instructions

Exemplary instructions for a kit and/or associated equipment andsolutions of the invention are set forth in this Example.

The Unstained High Molecular Weight (HMW) Protein Standard allowsaccurate molecular weight estimation of high molecular weight proteinson, e.g., NUPAGE® NOVEX® Tris-Acetate Gels with Tris-Acetate SDS buffersystem. A number of features of the standard are: it consists of 9protein bands in the range of 40-500 kDa; it is designed particularlybut not exclusively for use with NUPAGE® NOVEX® 3-8% and 7% Tris-AcetateGels under denaturing conditions; it is supplied in a ready-to-useformat; it is optionally visualized with Coomassie protein stain orsilver staining; and it is optionally visualized also with Ponceau S,Coomassie protein stain, or other membrane stains after westerntransfer.

In one embodiment, 250 ml of HIMARK™ Unstained High Molecular WeightProtein Standard is supplied in a storage buffer comprising 250 mMTris-HCl, pH 8.5; 0.5 mM EDTA; 50 mM DTT; 10% glycerol; 2% LDS; 0.2 mMCoomassie protein stain G-250; and 0.175 mM Phenol red, and stored at−20° C., under which conditions the Standard is stable for 6 months.Repeated freezing and thawing are avoided.

In one embodiment, the HIMARK™ Unstained HMW Protein Standard issupplied in a ready-to-use format. There is no need to heat or addreducing agent. The Standard is used as follows.

Thaw the standard at room temperature. Vortex gently to ensure thesolution is homogeneous. Note that if there is a precipitate in thestandard, thaw at room temperature for 10-15 minutes and vortex tosolubilize or warm the solution at 30° C. (do not heat at >37° C.).

Best results are obtained when 5 μl of Standard are loaded on a 1.0 mmthick Mini-Gel, or 7 μl are loaded on a 1.5 mm thick Mini-Gel. Use anappropriate percentage of NUPAGE® NOVEX® Tris-Acetate Gel to resolve theproteins.

After electrophoresis, gels are stained with SILVERQUEST® SilverStaining Kit, SIMPLYBLUE™ SafeStain, or Coomassie protein stain. Avoidusing SILVEREXPRESS® Silver Staining Kit.

When blotting gels, to obtain higher transfer efficiency during thetransfer of high molecular weight proteins, avoid using methanol in thetransfer buffer. After transfer, you may stain the standard proteins onthe membrane with Ponceau S, Coomassie or any membrane stain of choice.

Note that the HIMARK™ Unstained HMW Protein Standard is designed for usewith NUPAGE® NOVEX® Tris-Acetate Gels. Using the standards with NUPAGE®NOVEX® Bis-Tris Gels or Tris-Glycine Gels may result in inaccuratemolecular weight estimation.

In one embodiment, the HIMARK™ Unstained High Molecular Weight ProteinStandard is qualified on a NUPAGE® NOVEX® 3-8% Tris-Acetate Gel. Afterstaining with Coomassie R-250 stain, 9 sharp protein bands of theappropriate molecular weight are observed.

A molecular weight calculator for use with HIMARK™ High Molecular WeightProtein Standard is available for downloading on the worldwide web atinvitrogen.com by selecting the following links: Products &Services>Product Information>Life Science Products andServices>Electrophoresis>Protein Standards, Stains & Detection>ProteinStandards>HIJVIARK™ Standard for large protein analysis (40-500 kDa).The calculator provides an tool to easily and accurately calculate themolecular weight of your proteins on NUPAGE® NOVEX® 3-8% and 7%Tris-Acetate Gels and to extrapolate the molecular weight of proteinsbeyond the standard curve.

FIGS. 5A and 5B show Unstained HMW Protein Standard and a 460 kDaprotein run on gels. Unstained HMW Protein Standard (5 μl) and a 460 kDakinase were analyzed on a NUPAGE® NOVEX® 3-8% Tris-Acetate Gel (FIG. 5A)and a NUPAGE® NOVEX® 7% Tris-Acetate Gel (FIG. 5B), each of which iselectrophoresed in the presence of Tris-Acetate SDS buffer and stainedwith Coomassie R-250 stain. The apparent molecular weights of proteinstandard bands are shown in FIGS. 5A and 5B. The arrow indicates themigration of a 460 kDa kinase.

Example 8 De-Phosphorylation

In order to make a phosphorylated protein population homogeneous, theprotein is treated with kinase until the reaction is driven tocompletion to obtain a homogeneous population of phosphorylatedproteins. Alternatively, a population of phosphorylated proteins is madehomogeneous by dephosphorylation.

For example, for dephosphorylation, 100 μg of a phosphorylated orpartially phosphorylated protein sample is solubilized in 100 μl 50 mMTris/HCl, pH=7.5 containing 1 mM MgCl₂, and then incubated for about 10min at 30° C. Then, from 20 to about 30 units of calf intestine alkalinephosphatase (CIAP) are added (CIAP is commercially available from anumber of suppliers, including for example Stratagene, La Jolla, Calif.,and Promega, Madison, Wis.). The mixture is incubated for about 15minutes at about 30° C. The reaction is terminated by the addition of anequal volume of 2×SDS PAGE sample buffer and subsequent mixing. Thesample is analyzed by SDS-PAGE to confirm that the reaction is complete,nearly complete, complete to an acceptable extent or complete to adetectable limit.

Example 9 DNA Methylation

DNA is prepared according to standard techniques and treated with amethylase, preferably a methylase that recognizes a specific nucleotidesequence, such as a methylase of a restriction system. After themethylation reaction, the solution comprising DNA is digested with thecorresponding restriction enzyme to remove unmodified DNA molecules.

For example, a bacterial plasmid having a single EcoRI restriction sitehaving the following structure is prepared, preferably in supercoiledform:

The plasmid DNA is treated with EcoRI Methylase (New England Biolabs,Beverly, Mass.) essentially according to the manufacturer'sinstructions. In brief, reactions are carried out in 50 mM NaCl., 50 mMTris-HCl (pH 8.0), 10 mM EDTA, and 80 μM S-adenosylmethionine (SAM) isused as the methyl donor. The reactions are incubated at 37° C. BecauseEcoRI methylase is inhibited by MgCl₂, care is taken to avoid theaddition of MgCl₂ to the methylation reaction.

Following the methylation reaction, most if not all of the plasmids havemethyl residues to the EcoRI sites as indicated added as in thefollowing structure:

If need be, the resultant population of homogeneously methylated DNA isdigested with restriction enzyme EcoRI (New England Biolabs), anendonuclease that cleaves the GAATTC sequence only if theabove-described methylation has not taken place. The reaction is carriedout using an appropriate buffer such as 50 mM NaCl, 100 mM Tris-HCl, 10mM MgCl₂, 0.025% Triton X-100, pH 7.5). The reaction mixture isincubated at 37° C. The EcoRI enzyme is inactivated by heating at 65° C.for 20 minutes.

Digestion with the EcoRI endonuclease results in the linearization ofthe circular plasmid DNA. Methods for separating the undesired(unmethylated) linear DNA from the desired (methylated) circular plasmidDNA are known in the art and are used to further purify the methylatedDNA from other species of DNA. For example, DNA prepared from asupercoiled plasmid is separated from linear DNA by ultracentrifugationin a cesium chloride gradient in the presence of ethidium bromide. Theseparated plasmid DNA is a population of a species of DNA molecule thatis homogeneous, nearly homogeneous, homogeneous to a detectable limit,or homogeneous to a degree sufficient for the application.

The methylated circular DNA is then digested with a second restrictionenzyme, which may have one or more sites on the plasmid, in order togenerate a linear DNA standard, which may be molecular weight standardof the invention. The methylated plasmid can be one that comprises twoor more sites for the second restriction enzyme; if these sites are notequidistant from each other, digestion of the plasmid with the secondrestriction enzyme will yield two molecular species of different sizes,each of which is a homogeneous population. In this procedure, two ormore populations of homogeneous DNA molecules are prepared in a singletube.

Example 10 Pre-Stained Laminin

Deglycosylation, Reduction and Alkylation: Laminin is deglycosylatedessentially according to the method of Example 2 except thatconcentration by centrifugation is performed at 1800×g rather than3000×g. Laminin is then reduced and alkylated essentially according tothe method of Example 3, with the exceptions that the initial roomtemperature incubation is for 1.5 hours, concentration by centrifugationis performed at 1800×g, and 100 mM AMPSO (pH 9) is used in place of 50mM Tris (pH 8).

Staining: One ml of the 1 mg/ml deglycosylated, reduced and alkylatedlaminin solution, comprising 100 mM AMPSO (pH 9) and 1% SDS, is combinedwith 250 μl of 50 mg/ml Uniblue A in 20% SDS. The resulting sample isleft on a shaker for 30 minutes to solubilize the Uniblue A, and thenleft 16-20 hours at 37° C. to stain.

Purification: The resulting pre-stained laminin is purified by gelfiltration on TOYOPEARL® HM-40C resin (Tosoh Bioscience,Montgomeryville, Pa., USA) washed with 50 mM Tris (pH 8), 1% SDS. Thesame buffer solution (50 mM Tris (pH 8), 1% SDS) is used duringpurification.

Concentration: Purified pre-stained laminin is concentrated to 3 mg/mlusing an Amicon 100 kDa MWCO filter, with caution not to exceed the 3mg/ml target concentration.

Example 11 Pre-Stained β-Galactosidase

Alkylation: Alkylation is performed by mixing 1 ml of 1 mg/mlβ-galactosidase, prepared in 50 mM Tris (pH 8) and 2% SDS, with 10 μl of400 mM TBP in isopropyl alcohol. The sample is incubated at 70° C. for20 minutes, 100 μl of 1 M iodoacetic acid pH 7 in 25 mM phosphate bufferis added. The sample is further incubated at 70° C. for 10 minutes andat room temperature for 30 minutes.

Purification: The alkylated β-galactosidase is dialyzed overnightagainst 100 mM AMPSO (pH 9), 1% SDS. The sample is dialyzed 3 more hoursafter the dialysis buffer is changed (i.e. the old 100 mM AMPSO (pH 9),1% SDS is replaced with fresh).

Staining: Uniblue A (13 mg) is added to 1 ml of the purified alkylatedβ-galactosidase and the sample is incubated on a shaker for 1 hour atroom temperature, and then at 47° C. for 16-20 hours.

Purification: The resulting pre-stained β-galactosidase is purified bygel filtration on TOYOPEARL® HM-40C resin washed with 50 mM Tris (pH 8),1% SDS. The same buffer solution (50 mM Tris (pH 8), 1% SDS) is usedduring purification.

Concentration: Purified pre-stained β-galactosidase is concentrated to 2mg/ml using an Amicon 50 kDa MWCO filter.

Example 12 Himark™ Pre-Stained Standards

Pre-stained laminin and pre-stained β-galactosidase, prepared asdescribed in Examples 11 and 12, are mixed with other pre-stainedproteins to provide a mixture of pre-stained markers covering a broadrange of molecular weight. Pre-stained carbonic anhydrase, alcoholdehydrogenase, glutamic dehydrogenase and bovine serum albumin (MW) arecomponents of SeeBlue® Pre-Stained Standards (Catalog No. LC5625,Invitrogen, Carlsbad, Calif., USA). The pre-stained proteins mentionedin this Example may be mixed together to form the HIMARK™ Pre-StainedStandard. The amounts of the various pre-stained proteins are adjustedto create a mixture where the intensities of bands for the variousprotein markers are substantially similar, or reflect some desired ratioof intensities. Once the HIMARK™ Pre-Stained Standard is createdformamide sample buffer is added to 50% volume and it is stored at −20°C., at which temperature it does not freeze. Because the HIMARK™Pre-Stained Standard is not frozen there is no delay caused by the needto thaw the standard before use, and the standard does not undergorepeated freeze-thaw cycling.

Such pre-stained high molecular weight markers provide many advantages.The progress of electrophoretic runs can be assessed in real time byobserving the migration of pre-stained marker proteins as the runoccurs, so that a user can end the run at the time when optimalresolution has been achieved. The progress and efficiency of westerntransfer onto a membrane can also be monitored to know when, and if,transfer is complete. HIMARK™ Pre-Stained Standard provides theseadvantages to experiments involving high molecular weight proteins. Inaddition, pre-stained markers do not require a staining or otherdetection step after the gel is run, potentially simplifyingexperiments.

FIGS. 6-22 illustrate the use of the HIMARK™ Pre-Stained Standard. FIGS.6-9 illustrate the mobility of the HIMARK™ Pre-Stained Standard proteinsin various commonly used gels for separation of large proteins, as wellas the mobility of SEEBLUE® Plus2 Pre-Stained Standard proteins. Resultsare shown for 3-8% and 7% Tris-Acetate gels, a 4% Tris-Glycine gel and a4-12% NUPAGE® NOVEX® gel run with 1×MES. Unless otherwise indicated, allstandards in this Example are loaded at 10 μl/well.

FIGS. 10 and 11 illustrate the transfer of HIMARK™ Pre-Stained Standardproteins and SEEBLUE® Plus2 Pre-Stained Standard proteins tonitrocellulose membranes after electrophoresis.

FIGS. 12 and 13 illustrate the detection of proteins by western blot. InFIG. 12, 300 ng of large human protein, HIMARK™ Pre-Stained Standard andSEEBLUE® Plus2 Pre-Stained Standard are run on a 3-8% Tris-Acetate geland subsequently transferred to a nitrocellulose filter. Large humanprotein is detected using a specific polyclonal antibody. In FIG. 13, 5μl of BENCHMARK™ Unstained Standard, HIMARK™ Pre-Stained Standard andSEEBLUE® Plus2 Pre-Stained Standard are run on a 7% Tris-Acetate gel andsubsequently transferred to a nitrocellulose filter. BENCHMARK™Unstained Standard is detected using an anti-6×His antibody.

FIGS. 14 and 15 show results for experiments similar to those of FIGS.10 and 11, except that transfer is to PVDF rather than nitrocellulosemembranes.

FIGS. 16 and 17 show results for experiments similar to those of FIGS.12 and 13, except that transfer is to PVDF rather than nitrocellulosemembranes, and transfer is effected in the presence of 20% methanol andan antioxidant.

FIGS. 18 and 19 illustrate the transfer of HIMARK™ Pre-Stained Standardproteins and SEEBLUE® Plus2 Pre-Stained Standard proteins tonitrocellulose membranes after electrophoresis on a 4% Tris-Glycine gelor a 4-12% Bis-Tris gel run with MOPS, respectively.

FIG. 20 illustrates the use of HIMARK™ Pre-Stained Standard in a gelthat is stained with SIMPLYBLUE™ Safe Stain. Protein samples, including,from left to right, fibronectin, large human protein, human kinase andHIIMARK™ Pre-Stained Standard are run on a 3-8% Tris-Acetate gel andstained with SIMPLYBLUE™ Safe Stain.

FIGS. 21 and 22 illustrate the use of HIMARK™ Pre-Stained Standard ingels that are stained with fluorescent dyes, i.e. SYPRO® Orange andSYPRO® Ruby (Molecular Probes, Eugene, Oreg., USA), respectively.HIMARK™ Pre-Stained Standard proteins appear as dark bands on the darkgray backgrounds in FIGS. 21 and 22. Such staining and detection methodsmay be particularly advantageous when gel electrophoresis of a sample isto be followed by additional procedures, such as mass spectrometry.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitations,which is not specifically disclosed herein. The terms and expressionsthat have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed herein, optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims. In addition, where features or aspects of the inventionare described in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup of members of the Markush group. Theinvention has been described broadly and generically herein. Each of thenarrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. Other aspects ofthe invention are within the following claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference in their entirety to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference.

What is claimed is:
 1. A composition comprising a molecular weightprotein ladder comprising five or more purified proteins, each having adifferent apparent molecular weight by electrophoresis, wherein at leastone of the purified proteins is a first deglycosylated lamininpolypeptide having an apparent molecular weight greater than 200 kDa bySDS PAGE, wherein said first laminin polypeptide is optionally eitherreduced and alkylated, dephosphorylated, or dephosphorylated and reducedand alkylated; wherein said five or more purified proteins of themolecular weight protein ladder, when resolved and visualized on anelectrophoretic gel, display a pattern wherein the bands of the ladderare approximately equidistantly spaced from one another.
 2. Thecomposition according to claim 1, wherein said first laminin polypeptideis a laminin alpha chain, a laminin beta chain or a laminin gamma chain.3. The composition according to claim 1, wherein said first lamininpolypeptide has an apparent molecular weight selected from the listconsisting of 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950kDa, or 1000 kDa.
 4. The composition according to claim 1, wherein saidfirst laminin polypeptide is a laminin alpha protein having molecularweight of 425-525 kDa, 475-525, or 500 kDa, or wherein said firstlaminin polypeptide is a laminin beta protein having an estimatedmolecular weight of 200-375 kDa, 250-325 kDa, or 290 kDa, or whereinsaid first laminin polypeptide is a laminin gamma protein having anestimated molecular weight of 200-300 kDa, 225-275 kDa, or 240 kDa. 5.The composition according to claim 1, further comprising a secondlaminin polypeptide, wherein said second laminin polypeptide has adifferent molecular weight from the first laminin polypeptide.
 6. Thecomposition according to claim 5, wherein the second laminin polypeptidehas an apparent molecular weight of 250 kDa, 300 kDa, 350 kDa, 400 kDa,450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa,850 kDa, 900 kDa, 950 kDa, or 1000 kDa.
 7. The composition according toclaim 1, wherein at least two of said purified proteins have an apparentmolecular weight of greater than 300 kDa.
 8. The composition accordingto claim 1, wherein at least one of said purified proteins ispre-stained.
 9. The composition according to claim 1, wherein thepopulation of proteins comprises three laminin polypeptides.
 10. Thecomposition according to claim 9, wherein at least two of the lamininpolypeptides are modified by deglycosylation and optionally alkylation,and optionally wherein at least one of the phosphate groups of the oneor more of the laminin polypeptides are removed.
 11. The compositionaccording to claim 1, wherein said first laminin polypeptide is reducedand alkylated.